U.S. patent application number 17/558731 was filed with the patent office on 2022-04-14 for method of treating cancer using selective estrogen receptor modulators.
The applicant listed for this patent is Duke University. Invention is credited to Donald P McDonnell, Erik R. Nelson, Suzanne E. Wardell.
Application Number | 20220110893 17/558731 |
Document ID | / |
Family ID | |
Filed Date | 2022-04-14 |
View All Diagrams
United States Patent
Application |
20220110893 |
Kind Code |
A1 |
Wardell; Suzanne E. ; et
al. |
April 14, 2022 |
METHOD OF TREATING CANCER USING SELECTIVE ESTROGEN RECEPTOR
MODULATORS
Abstract
Disclosed herein are methods of treating subjects suffering from
estrogen receptor positive cancer of the brain by administering a
selective estrogen receptor degrader (SERM). Also disclosed are
methods of treating a cancer that is resistant to an estrogen
receptor modulator by administering a SERM.
Inventors: |
Wardell; Suzanne E.;
(Durham, NC) ; Nelson; Erik R.; (Champaign,
IL) ; McDonnell; Donald P; (Chapel Hill, NC) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Duke University |
Durham |
NC |
US |
|
|
Appl. No.: |
17/558731 |
Filed: |
December 22, 2021 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
16549828 |
Aug 23, 2019 |
|
|
|
17558731 |
|
|
|
|
15129197 |
Sep 26, 2016 |
10420734 |
|
|
PCT/US2015/023216 |
Mar 28, 2015 |
|
|
|
16549828 |
|
|
|
|
14512061 |
Oct 10, 2014 |
9421264 |
|
|
15129197 |
|
|
|
|
62129379 |
Mar 6, 2015 |
|
|
|
61971627 |
Mar 28, 2014 |
|
|
|
61971627 |
Mar 28, 2014 |
|
|
|
International
Class: |
A61K 31/137 20060101
A61K031/137; A61K 9/00 20060101 A61K009/00; A61K 31/136 20060101
A61K031/136; A61K 31/138 20060101 A61K031/138; A61K 31/40 20060101
A61K031/40; A61K 31/4196 20060101 A61K031/4196; A61K 31/4535
20060101 A61K031/4535; A61K 31/565 20060101 A61K031/565; A61K
31/5685 20060101 A61K031/5685; A61K 45/06 20060101 A61K045/06; C07C
217/78 20060101 C07C217/78; C07C 217/84 20060101 C07C217/84 |
Goverment Interests
STATEMENT OF GOVERNMENT INTEREST
[0002] This invention was made with government support under
R37DK048807 awarded by the National Institutes of Health. The
government has certain rights in the invention.
Claims
1. A method of treating an estrogen receptor positive breast cancer
in a subject, wherein the estrogen receptor positive breast cancer
is resistant to an estrogen receptor modulator, the method
comprising administering a composition comprising a compound
represented by the following formula I: ##STR00022## wherein TZ
represents a C.sub.1-C.sub.4 alkylene group or
--CR.sup.f'R.sup.g'--CH.sub.2--O-- wherein R.sup.f' and R.sup.g'
independently represent hydrogen or a C.sub.1-C.sub.6 alkyl group;
A represents a 5- to 14-membered heteroarylene group which may have
a substituent or a C.sub.6-C.sub.14 arylene group which may have a
substituent; Y represents --CH.sub.2--NR.sup.c-- wherein R.sup.c
represents hydrogen or a C.sub.1-C.sub.6 alkyl group which may have
a substituent; ring G represents the following formula:
##STR00023## R' represents 1 to 4 substituents independently
selected from a hydrogen atom, a C.sub.1-C.sub.6 alkoxy group, and
a hydroxyl group; a partial structure in formula (I) represented by
the following formula: ##STR00024## R'' represents hydrogen, a
hydroxyl group that may be further protected by a protecting group
or a C.sub.1-C.sub.6 alkoxy group which may have a substituent;
R.sup.a and R.sup.b are the same as or different from each other
and each represents a hydrogen atom, a C.sub.1-C.sub.6 alkyl group
which may have a substituent, or a C.sub.3-C.sub.8 cycloalkyl group
which may have a substituent, or when R.sup.a and R.sup.b are
bonded together, they may form, together with the nitrogen atom
that is adjacent to R.sup.a and R.sup.b, a 4- to 10-membered single
ring which may have a substituent; and L represents a single
bond.
2. The method of claim 1, wherein the estrogen receptor positive
breast cancer is de novo resistant to the estrogen receptor
modulator.
3. The method of claim 1, wherein the resistance to the estrogen
receptor modulator is acquired.
4. The method of claim 1, wherein the estrogen receptor modulator
is a selective estrogen receptor modulator (SERM).
5. The method of 4, wherein the SERM is tamoxifen, idoxifene,
raloxifene or ICI 182,780.
6. The method of claim 1, wherein R.sup.a and R.sup.b independently
represent a hydrogen atom, a methyl group, an ethyl group, a
n-propyl group, an iso-propyl group, a n-butyl group, an iso-butyl
group, or a tert-butyl group.
7. The method of claim 1, wherein -T-Z-- represents
--CH.sub.2CH.sub.2-- or --C(CH.sub.3).sub.2CH.sub.2O--.
8. The method of claim 1, wherein Y represents
--CH.sub.2--N(CH.sub.2CH.sub.3)-- or
--CH.sub.2--N(CH.sub.2CH.sub.2OH)--.
9. The method of claim 1, wherein each of R' independently
represents a hydrogen atom or a methoxy group.
10. The method of claim 1, wherein R'' represents a hydroxyl
group.
11. The method of claim 1, wherein A represents a phenylene
group.
12. The method of claim 1, wherein an effective amount of the
compound is administered.
13. The method of claim 12, wherein the effective amount comprises
a high dosage.
14. The method of claim 1, wherein the compound is administered by
oral administration, intravenous administration, intradermal
injection, intramuscular injection, or subcutaneous injection.
15. The method of claim 1, further comprising administering an
effective amount of at least one compound selected from the group
consisting of a cyclin-dependent kinase 4 and 6 inhibitor (CDK4/6
inhibitor), an antiestrogen, a ligand of retinoic acid or retinoxic
X receptor, an antiprogestin, an antiandrogen, vitamin D or
metabolite thereof, a farnesyl transferase inhibitor, a PPAR.alpha.
or gamma agonist and a MAP kinase inhibitor.
16. The method of claim 13, wherein the high dosage is more than
about 20 mg/kg.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This patent application is a continuation U.S. patent
application Ser. No. 16/549,828, filed Aug. 23, 2019, which is a
continuation of U.S. patent application Ser. No. 15/129,197, filed
Sep. 26, 2016, issued Sep. 24, 2019 as U.S. Pat. No. 10,420,734,
which is the national stage filing under 35 U.S.C. .sctn. 371 of
International Application No. PCT/US2015/023216, filed on Mar. 28,
2015, which application claims priority to U.S. Provisional
Application No. 62/129,379, filed Mar. 6, 2015, and is a
continuation-in-part of U.S. patent application Ser. No.
14/512,061, filed Oct. 10, 2014, issued as U.S. Pat. No. 9,421,264,
on Aug. 23, 2016, which application claims priority to U.S.
Provisional Application No. 61/971,627, filed Mar. 28, 2014, the
entire contents of each of which are hereby incorporated by
reference.
TECHNICAL FIELD
[0003] The present invention relates to methods of treating
subjects suffering from estrogen receptor positive cancer of the
brain by administering a selective estrogen receptor modulator
(SERM) to the subject. The present invention also relates to
methods of treating subjects suffering from a cancer that is
resistant to an estrogen receptor modulator by administering a SERM
to the subject.
BACKGROUND
[0004] The estrogen receptor (ER) is a ligand dependent
transcription factor whose expression confers upon target cells the
ability to respond to estrogens. In the absence of an activating
ligand, ER resides in the cell in an inactive form within a large
inhibitory protein complex. Upon binding ligand, however, the
receptor undergoes an activating conformational change resulting in
its release from the inhibitory protein complex, spontaneous
dimerization and subsequent interaction with enhancers located
within target genes. Depending on the promoter context of the bound
receptor, and the co factors that are recruited to the receptor in
a particular cell, it can either positively or negatively regulate
target gene transcription. Thus, the same ER-ligand complex can
have very different activities in different cells, an observation
that explains how estrogens, generally considered to be
reproductive hormones, exhibit activities in bone, the
cardiovascular system and in brain that are unrelated to
reproductive function.
[0005] Whereas the molecular determinants of ER action differ
considerably between target cells, it has been anticipated that the
exploitation of this complexity will yield pharmaceuticals with
process or tissue selective activities. The first evidence in
support of this hypothesis came from studies that probed the
pharmacological activities of the `antiestrogen` tamoxifen.
Identified as a high affinity antagonist of ER and developed as a
treatment for ER-positive breast cancer, it soon became apparent
that whereas tamoxifen could oppose estrogen action in the breast
it exhibited agonist activity in the bone, uterus and in the
cardiovascular system. Reflecting this spectrum of activities,
tamoxifen was reclassified as a Selective Estrogen Receptor
Modulator (SERM).
[0006] Breast cancer remains the most commonly diagnosed cancer
among women and a leading cause of cancer mortality. While targeted
therapies such as the SERM tamoxifen and aromatase inhibitors are
initially effective in the treatment of estrogen receptor alpha
(ESR1) positive tumors, de novo and acquired resistance remain an
impediment to durable clinical responses, particularly in the
setting of advanced disease. ESR1 is a therapeutic target in breast
cancers that are resistant to both first and second line endocrine
interventions, a finding that has prompted the development of (a)
SERMs with a mechanism distinct from tamoxifen and (b) selective
estrogen receptor degraders (SERDs), competitive antagonists whose
interaction with ESR1 induce its proteasome dependent degradation.
Fulvestrant, a SERD, has been effective as both a first- and
second-line therapy in advanced breast cancer; however, the
pharmaceutical properties of this drug may prove dose-limiting in
relapsed/resistant breast tumors bearing ESR1 mutations known to
decrease SERD potency. The clinical efficacy of fulvestrant, a
Selective Estrogen Receptor Degrader (SERD) that triggers receptor
degradation, has confirmed that ESR1 often remains engaged in
endocrine therapy resistant cancers.
[0007] The increasing incidence of breast cancer brain metastases
(BCBM) is an emerging challenge in the treatment of advanced breast
cancer patients. The growing success of improved treatments of
systemic disease has allowed the manifestation of BCBM that
previously would not have impacted the morbidity and mortality
associated with breast cancer. The privileged environment of the
brain, maintained by the relatively non-porous blood brain barrier,
presents a significant impediment to the successful targeting of
BCBM, leading to the use of gamma knife surgery and/or whole brain
radiation in an attempt to shrink or ablate brain lesions. The
benefit of these treatments must be carefully balanced with
neurological deficit as a result of treatment.
[0008] Although considerable advances have been made in targeting
the estrogen signaling axis for the treatment of breast cancer and
osteoporosis, similar progress has unfortunately not yet been
accomplished in the development of safe and effective treatments
for the climacteric conditions or vasomotor disturbances that are
associated with estrogen deprivation. There is considerable
interest in developing novel SERMs that can be used to treat
vasomotor symptoms but which do not exhibit mitogenic activities in
the breast or the uterus.
[0009] While tamoxifen and aromatase inhibitors have proven
effective in the treatment of estrogen receptor positive (ER+)
breast cancer, the incidence of resistance remains significant,
particularly in the advanced/metastatic breast cancer setting. An
additional class of estrogen receptor targeting therapy, selective
estrogen receptor degraders (SERDs), has recently come to
prominence. These agents have proven effective in pre-clinical
models of breast cancers that are resistant to tamoxifen or
aromatase inhibitors, leading to their evaluation in clinical
trials. However, these agents also do not readily pass the blood
brain barrier, suggesting that they will be ineffective in
targeting BCBM. It would be beneficial to have other treatment
options that can penetrate the blood brain barrier and/or
selectively target tissue specific activities responsive to ER
activation.
SUMMARY
[0010] The present invention is directed to a method of treating
estrogen receptor positive cancers of the brain in a subject. The
method comprises administering a compound represented by the
following formula I:
##STR00001##
wherein [0011] TZ represents a C.sub.1-C.sub.4 alkylene group or
--CR.sup.f'R.sup.g'--CH.sub.2--O-- wherein R.sup.f' and R.sup.g'
independently represent hydrogen or a C1-C6 alkyl group; [0012] A
represents a 5- to 14-membered heteroarylene group which may have a
substituent or a C.sub.6-C.sub.14 arylene group which may have a
substituent; [0013] Y represents --CH.sub.2--NR.sup.c-- wherein
R.sup.c represents hydrogen or a C.sub.1-C.sub.6 alkyl group which
may have a substituent; [0014] ring G represents the following
formula:
[0014] ##STR00002## [0015] R' represents 1 to 4 substituents
independently selected from a hydrogen atom, a C.sub.1-C.sub.6
alkoxy group, and a hydroxyl group; [0016] a partial structure in
formula (I) represented by the following formula:
[0016] ##STR00003## [0017] R'' represents hydrogen, a hydroxyl
group that may be further protected by a protecting group or a
C.sub.1-C.sub.6 alkoxy group which may have a substituent; and
[0018] R.sup.a and R.sup.b are the same as or different from each
other and each represents a hydrogen atom, a C.sub.1-C.sub.6 alkyl
group which may have a substituent, or a C.sub.3-C.sub.8 cycloalkyl
group which may have a substituent, or when R.sup.a and R.sup.b are
bonded together, they may form, together with the nitrogen atom
that is adjacent to R.sup.a and R.sup.b, a 4- to 10-membered single
ring which may have a substituent; and [0019] L represents a single
bond, or a salt thereof.
[0020] The cancer may be Breast cancer brain metastases,
Astrocytoma, Atypical Teratoid Rhabdoid Tumor (ATRT),
Chondrosarcoma, Choroid Plexus Carcinoma, Craniopharyngioma,
Ependymoma, Germ Cell Tumor, Glioblastoma, Glioma, Hemangioma,
Juvenile Pilocytic Astrocytoma, Medulloblastoma, Meningioma,
Neurofibroma, Neuronal and Mixed Neuronal-Glial Tumors,
Oligoastrocytoma, Oligodendroglioma, Pineal Tumor, Pituitary Tumor,
PNET--(primitive neuroectodermal tumor), Schwannoma, and
Leptomeningeal metastases. Ra and Rb independently may represent a
hydrogen atom, a methyl group, an ethyl group, a n-propyl group, an
iso-propyl group, a n-butyl group, an iso-butyl group, or a
tert-butyl group. -T-Z-- may represents --CH.sub.2CH.sub.2-- or
--C(CH.sub.3).sub.2CH.sub.2O--. Y may represent
--CH.sub.2--N(CH.sub.2CH.sub.3)-- or
--CH.sub.2--N(CH.sub.2CH.sub.2OH)--. Each of R'' independently may
represents a hydrogen atom or a methoxy group. R'' may represents a
hydroxyl group. A may represents a phenylene group. The compound
may be
(R)-6-{2-{ethyl[4-(2-ethylaminoethyl)benzyl]amino}-4-methoxyphenyl}-5,6,7-
,8-tetrahydronaphthalen-2-ol. An effective amount of the compound
may be administered. The effective amount may comprise a high
dosage. The high dosage may be more than about 20 mg/kg. The high
dosage may be about 20 mg/kg to about 100 mg/kg. The compound may
be administered by oral administration, intravenous administration,
intradermal injection, intramuscular injection, or subcutaneous
injection. The method may further comprising administering an
effective amount of at least one compound selected from the group
consisting of a cyclin-dependent kinase 4 and 6 inhibitor (CDK4/6
inhibitor), an antiestrogen, a ligand of retinoic acid or retinoxic
X receptor, an antiprogestin, an antiandrogen, vitamin D or
metabolite thereof, a farnesyl transferase inhibitor, a PPAR.alpha.
or gamma agonist and a MAP kinase inhibitor.
[0021] The present invention is directed to a method of treating
breast cancer brain metastasis in a subject. The method comprises
administering a compound represented by the following formula
I:
##STR00004##
wherein [0022] TZ represents a C.sub.1-C.sub.4 alkylene group or
--CR.sup.f'R.sup.g'--CH.sub.2--O-- wherein R.sup.f' and R.sup.g'
independently represent hydrogen or a C.sub.1-C.sub.6 alkyl group;
[0023] A represents a 5- to 14-membered heteroarylene group which
may have a substituent or a C.sub.6-C.sub.14 arylene group which
may have a substituent; [0024] Y represents --CH.sub.2--NR.sup.c--
wherein R.sup.c represents hydrogen or a C.sub.1-C.sub.6 alkyl
group which may have a substituent; [0025] ring G represents the
following formula:
[0025] ##STR00005## [0026] R' represents 1 to 4 substituents
independently selected from a hydrogen atom, a C.sub.1-C.sub.6
alkoxy group, and a hydroxyl group; [0027] a partial structure in
formula (I) represented by the following formula:
[0027] ##STR00006## [0028] R'' represents hydrogen, a hydroxyl
group that may be further protected by a protecting group or a
C.sub.1-C.sub.6 alkoxy group which may have a substituent; and
[0029] R.sup.a and R.sup.b are the same as or different from each
other and each represents a hydrogen atom, a C.sub.1-C.sub.6 alkyl
group which may have a substituent, or a C.sub.3-C.sub.8 cycloalkyl
group which may have a substituent, or when R.sup.a and R.sup.b are
bonded together, they may form, together with the nitrogen atom
that is adjacent to R.sup.a and R.sup.b, a 4- to 10-membered single
ring which may have a substituent; and [0030] L represents a single
bond, or a salt thereof.
[0031] R.sup.a and R.sup.b independently may represent a hydrogen
atom, a methyl group, an ethyl group, a n-propyl group, an
iso-propyl group, a n-butyl group, an iso-butyl group, or a
tert-butyl group. -T-Z-- may represents --CH.sub.2CH.sub.2-- or
--C(CH.sub.3).sub.2CH.sub.2O--. Y may represent
--CH.sub.2--N(CH.sub.2CH.sub.3)-- or
--CH.sub.2--N(CH.sub.2CH.sub.2OH)--. Each of R'' independently may
represents a hydrogen atom or a methoxy group. R'' may represents a
hydroxyl group. A may represents a phenylene group. The compound
may be
(R)-6-{2-{ethyl[4-(2-ethylaminoethyl)benzyl]amino}-4-methoxyphenyl}-5,6,7-
,8-tetrahydronaphthalen-2-ol. An effective amount of the compound
may be administered. The effective amount may comprise a high
dosage. The high dosage may be more than about 20 mg/kg. The high
dosage may be about 20 mg/kg to about 100 mg/kg. The compound may
be administered by oral administration, intravenous administration,
intradermal injection, intramuscular injection, or subcutaneous
injection. The method may further comprising administering an
effective amount of at least one compound selected from the group
consisting of a cyclin-dependent kinase 4 and 6 inhibitor (CDK4/6
inhibitor), an antiestrogen, a ligand of retinoic acid or retinoxic
X receptor, an antiprogestin, an antiandrogen, vitamin D or
metabolite thereof, a farnesyl transferase inhibitor, a PPAR.alpha.
or gamma agonist and a MAP kinase inhibitor.
[0032] The present invention is directed to a method of treating a
cancer in a subject, wherein the cancer is resistant to an estrogen
receptor modulator. The method comprises administering a compound
represented by the following formula I:
##STR00007##
wherein [0033] TZ represents a C.sub.1-C.sub.4 alkylene group or
--CR.sup.f'R.sup.g'--CH.sub.2--O-- wherein R.sup.f' and R.sup.g'
independently represent hydrogen or a C.sub.1-C.sub.6 alkyl group;
[0034] A represents a 5- to 14-membered heteroarylene group which
may have a substituent or a C.sub.6-C.sub.14 arylene group which
may have a substituent; [0035] Y represents --CH.sub.2--NR.sup.c--
wherein R.sup.c represents hydrogen or a C.sub.1-C.sub.6 alkyl
group which may have a substituent; [0036] ring G represents the
following formula:
[0036] ##STR00008## [0037] R' represents 1 to 4 substituents
independently selected from a hydrogen atom, a C.sub.1-C.sub.6
alkoxy group, and a hydroxyl group; [0038] a partial structure in
formula (I) represented by the following formula:
[0038] ##STR00009## [0039] R'' represents hydrogen, a hydroxyl
group that may be further protected by a protecting group or a
C.sub.1-C.sub.6 alkoxy group which may have a substituent; and
[0040] R.sup.a and R.sup.b are the same as or different from each
other and each represents a hydrogen atom, a C.sub.1-C.sub.6 alkyl
group which may have a substituent, or a C.sub.3-C.sub.8 cycloalkyl
group which may have a substituent, or when R.sup.a and R.sup.b are
bonded together, they may form, together with the nitrogen atom
that is adjacent to R.sup.a and R.sup.b, a 4- to 10-membered single
ring which may have a substituent; and [0041] L represents a single
bond, or a salt thereof.
[0042] The cancer may be de novo resistant to the estrogen receptor
modulator. The resistance to the estrogen receptor modulator may be
acquired. The estrogen receptor modulator may be a selective
estrogen receptor modulator (SERM). The SERM may be tamoxifen,
idoxifene, raloxifene or ICI 182,780. The cancer may be breast,
endometrial or ovarian cancer. The cancer may be breast cancer.
R.sup.a and R.sup.b independently may represent a hydrogen atom, a
methyl group, an ethyl group, a n-propyl group, an iso-propyl
group, a n-butyl group, an iso-butyl group, or a tert-butyl group.
-T-Z-- may represents --CH.sub.2CH.sub.2-- or
--C(CH.sub.3).sub.2CH.sub.2O--. Y may represent
--CH.sub.2--N(CH.sub.2CH.sub.3)-- or
--CH.sub.2--N(CH.sub.2CH.sub.2OH)--. Each of R'' independently may
represents a hydrogen atom or a methoxy group. R'' may represents a
hydroxyl group. A may represents a phenylene group. The compound
may be
(R)-6-{2-{ethyl[4-(2-ethylaminoethyl)benzyl]amino}-4-methoxyphenyl}-5,6,7-
,8-tetrahydronaphthalen-2-ol. An effective amount of the compound
may be administered. The effective amount may comprise a high
dosage. The high dosage may be more than about 20 mg/kg. The high
dosage may be about 20 mg/kg to about 100 mg/kg. The compound may
be administered by oral administration, intravenous administration,
intradermal injection, intramuscular injection, or subcutaneous
injection. The method may further comprising administering an
effective amount of at least one compound selected from the group
consisting of a cyclin-dependent kinase 4 and 6 inhibitor (CDK4/6
inhibitor), an antiestrogen, a ligand of retinoic acid or retinoxic
X receptor, an antiprogestin, an antiandrogen, vitamin D or
metabolite thereof, a farnesyl transferase inhibitor, a PPAR.alpha.
or gamma agonist and a MAP kinase inhibitor.
BRIEF DESCRIPTION OF THE DRAWINGS
[0043] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee.
[0044] FIGS. 1A-1B show treatment with
(R)-6-{2-{ethyl[4-(2-ethylaminoethyl)benzyl]amino}-4-methoxyphenyl}-5,6,7-
,8-tetrahydronaphthalen-2-ol ("RAD1901") results in a dose
dependent reduction in ER expression and activity. FIG. 1A shows
MCF7 breast cancer cells were treated 4 hours as indicated prior to
western blot detection of ER and loading control cytokeratin (CK)
18. FIG. 1B shows ovariectomized mice bearing MCF7 xenograft tumors
were treated daily with RAD1901 ("RAD") or tamoxifen ("Tam") in the
context of continued estrogen treatment.
[0045] FIGS. 2A-2B show the mechanism by which RAD1901
downregulates ER expression. FIG. 2A shows ER.alpha. protein
expression in whole cell extracts pre-treated with transcription or
translation inhibitors before treatment with RAD1901, analyzed by
immunoblot. FIG. 2B shows ER.alpha. mRNA expression in similarly
treated cells.
[0046] FIG. 3 shows the interaction between ER and
conformation-specific peptides in mammalian two-hybrid system.
[0047] FIGS. 4A-4B show RAD1901 exhibits dose dependent
agonist/antagonist regulation of ER transactivation of target
genes.
[0048] FIGS. 5A-5F show that RAD1901 inhibited ESR1 activity in
vitro and in vivo.
[0049] FIGS. 6A-6D show that RAD1901 downregulated ESR1 expression
through receptor degradation.
[0050] FIGS. 7A-7D show that the effects of RAD1901 on BT483 breast
cancer cells were similar to those observed in MCF7 cells.
[0051] FIGS. 8A-8C show that the in vivo pharmacology of RAD1901
was influenced by SERD activity.
[0052] FIGS. 9A-9E show that RAD1901 exhibited dose dependent
growth stimulation of MCF7 xenograft tumors.
[0053] FIGS. 10A-10E show that RAD1901 exerted biphasic
agonist/antagonist activity on ESR1 in a dose dependent manner.
[0054] FIG. 11 shows the relative change in expression of ESR1
target genes and additional target genes responsive to agonists,
primarily SERMs, or SERMs and agonists.
DETAILED DESCRIPTION
[0055] The present disclosure provides a method of treating a
subject suffering from estrogen receptor positive cancer of the
brain, such as BCBM, or a cancer that is resistant to an estrogen
receptor modulator, such as tamoxifen resistant breast cancer. The
methods involve administering to the subject a SERM, such as
RAD1901. RAD1901 is a SERM/SERD hybrid that exhibits complex and
unique pharmacology in breast cancer models, having dose-dependent
agonist/antagonist activity displayed in a tissue-selective manner.
RAD1901 exhibits desired pharmacological activities and exhibits
significant brain penetrance when evaluated in postmenopausal
women, in particular a unique dose response, with lower doses of
the drug being more effective at relieving hot flashes. RAD1901
induces hot flashes in healthy postmenopausal women in a dose
dependent manner, thus RAD1901 may effectively inhibit estrogen
receptor action in the brain.
[0056] Turnover of ER.alpha. is significantly increased upon
binding RAD1901, an activity that is more pronounced at higher drug
concentrations (FIG. 1). This drug exhibits some of the
characteristics that are generally attributed to selective estrogen
receptor degraders (SERDs). Thus, at lower doses RAD1901 exhibits
partial agonist activity, i.e., SERM activity, allowing for relief
of hot flashes, but the SERD activity of the compound dominates
when the receptor is exposed to higher concentrations. The present
disclosure describes in vitro the mechanism by which RAD1901
impacts ER expression and investigates the possible result of such
action in vivo.
[0057] As exemplified below, RAD1901 surprisingly has the ability
to degrade the estrogen receptor. Both in vitro and in vivo studies
have since shown that the antagonist activity of this ligand
correlates with estrogen receptor degradation in a dose dependent
manner. RAD1901 also inhibits estrogen dependent growth of breast
cancer xenograft tumors and may be used to treat breast cancer,
such as tamoxifen resistant breast cancer.
[0058] RAD1901 is unique among both SERMs and SERDs in that this
drug accumulates in the brain, an environment in which SERM
penetration has been historically regarded as quite low. Estrogen
receptor activity has been found to be important in the growth of
tumors resistant to aromatase inhibitors and/or tamoxifen, and
treatment with SERDs has been shown to have clinical benefit. While
the revelation that RAD1901 exhibits SERD activity certainly
suggests potential utility in the treatment of progressing ER+
breast tumors, which was unappreciated prior to the present
disclosure, the targeting of ER activity for the treatment of ER+
brain cancers represents a new frontier for the use of both SERMs
and SERDs, as RAD1901 represents the first SERM/SERD that can
sufficiently penetrate the brain to exhibit efficacy. Because BCBM
is generally diagnosed late in the disease progression of ER+
metastatic breast cancer, patients will in general have already
been treated with endocrine therapeutics (i.e. tamoxifen or
aromatase inhibitors). Thus, while SERMs such as tamoxifen have low
brain penetration and have exhibited efficacy in anecdotal cases of
BCBM that are detailed in the literature, the SERD activity of
RAD1901 becomes key to the therapeutic potential of this compound
for treatment of BCBM, as SERDs have been found to be effective in
breast cancers that are resistant to SERM or aromatase inhibitor
therapy.
1. Definitions
[0059] The terms "comprise(s)," "include(s)," "having," "has,"
"can," "contain(s)," and variants thereof, as used herein, are
intended to be open-ended transitional phrases, terms, or words
that do not preclude the possibility of additional acts or
structures. The singular forms "a," "and" and "the" include plural
references unless the context clearly dictates otherwise. The
present disclosure also contemplates other embodiments
"comprising," "consisting of" and "consisting essentially of," the
embodiments or elements presented herein, whether explicitly set
forth or not.
[0060] For the recitation of numeric ranges herein, each
intervening number there between with the same degree of precision
is explicitly contemplated. For example, for the range of 6-9, the
numbers 7 and 8 are contemplated in addition to 6 and 9, and for
the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6,
6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.
[0061] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art. In case of conflict, the present
document, including definitions, will control. Preferred methods
and materials are described below, although methods and materials
similar or equivalent to those described herein can be used in
practice or testing of the present invention. All publications,
patent applications, patents and other references mentioned herein
are incorporated by reference in their entirety. The materials,
methods, and examples disclosed herein are illustrative only and
not intended to be limiting.
[0062] The term "administration" or "administering," as used herein
refers to providing, contacting, and/or delivery of the SERM by any
appropriate route to achieve the desired effect. These agents may
be administered to a subject in numerous ways including, but not
limited to, orally, ocularly, nasally, intravenously, topically, as
aerosols, suppository, etc. and may be used in combination.
[0063] "Aromatase inhibitor" as used herein refers to a compound
that targets aromatase, which is an enzyme involved in the
biosynthesis of estrogen. Aromatase inhibitors may block the
production of estrogen or block the action of estrogen on
receptors.
[0064] "Blood brain barrier" or "BBB" as used herein refers to a
highly selective permeability barrier that separates the
circulating blood from the brain extracellular fluid in the central
nervous system. The blood brain barrier may prevent the certain
drugs from entering brain tissue and is a limiting factor in the
delivery of many peripherally-administered agents to the central
nervous system.
[0065] "Breast cancer" as used herein refers to a type of cancer
that originates from and develops in the breast. "Metastatic breast
cancer" refers to breast cancer that spreads outside the breast to
the lymph nodes, bones, or other areas.
[0066] "Breast Cancer Brain Metastases" and "BCBM" as used
interchangeably herein refer to breast cancer that has metastasized
to the brain. BCBM may occur in up to 10-15% of breast-cancer
patients. BCBM may progress rapidly and can produce
life-threatening complications such as increased intracranial
pressure, herniation of the brain and seizures. Radiotherapy is a
treatment of BCBM as it halts tumor progression quickly and can
induce a response in the majority of patients.
[0067] "Cancer" as used herein refers to the uncontrolled and
unregulated growth of abnormal cells in the body. Cancerous cells
are also called malignant cells. Cancer may invade nearby parts of
the body and may also spread to more distant parts of the body
through the lymphatic system or bloodstream. Cancers include
Adrenocortical Carcinoma, Anal Cancer, Bladder Cancer, Brain Tumor,
Breast Cancer, Carcinoid Tumor, Gastrointestinal, Carcinoma of
Unknown Primary, Cervical Cancer, Colon Cancer, Endometrial Cancer,
Esophageal Cancer, Extrahepatic Bile Duct Cancer, Ewings Family of
Tumors (PNET), Extracranial Germ Cell Tumor, Intraocular Melanoma
Eye Cancer, Gallbladder Cancer, Gastric Cancer (Stomach),
Extragonadal Germ Cell Tumor, Gestational Trophoblastic Tumor, Head
and Neck Cancer, Hypopharyngeal Cancer, Islet Cell Carcinoma,
Kidney Cancer (renal cell cancer), Laryngeal Cancer, Acute
Lymphoblastic Leukemia, Leukemia, Acute Myeloid, Chronic
Lymphocytic Leukemia, Chronic Myelogenous Leukemia, Hairy Cell
Leukemia, Lip and Oral Cavity Cancer, Liver Cancer, Non-Small Cell
Lung Cancer, Small Cell Lung Cancer, AIDS-Related Lymphoma, Central
Nervous System (Primary) Lymphoma, Cutaneous T-Cell Lymphoma,
Hodgkin's Disease Lymphoma, Non-Hodgkin's Disease Lymphoma,
Malignant Mesothelioma, Melanoma, Merkel Cell Carcinoma, Metasatic
Squamous Neck Cancer with Occult Primary, Multiple Myeloma and
Other Plasma Cell Neoplasms, Mycosis Fungoides, Myelodysplastic
Syndrome, Myeloproliferative Disorders, Nasopharyngeal Cancer,
euroblastoma, Oral Cancer, Oropharyngeal Cancer, Osteosarcoma,
Ovarian Epithelial Cancer, Ovarian Germ Cell Tumor, Pancreatic
Cancer, Exocrine, Pancreatic Cancer, Islet Cell Carcinoma,
Paranasal Sinus and Nasal Cavity Cancer, Parathyroid Cancer, Penile
Cancer, Pituitary Cancer, Plasma Cell Neoplasm, Prostate Cancer,
Rhabdomyosarcoma, Rectal Cancer, Renal Cell Cancer (cancer of the
kidney), Transitional Cell Renal Pelvis and Ureter, Salivary Gland
Cancer, Sezary Syndrome, Skin Cancer, Small Intestine Cancer, Soft
Tissue Sarcoma, Testicular Cancer, Malignant Thymoma, Thyroid
Cancer, Urethral Cancer, Uterine Cancer, Unusual Cancer of
Childhood, Vaginal Cancer, Vulvar Cancer, and Wilms' Tumor.
[0068] The term "effective dosage" as used herein means a dosage of
a drug effective for periods of time necessary, to achieve the
desired therapeutic result. An effective dosage may be determined
by a person skilled in the art and may vary according to factors
such as the disease state, age, sex, and weight of the individual,
and the ability of the drug to elicit a desired response in the
individual. This term as used herein may also refer to an amount
effective at bringing about a desired in vivo effect in an animal,
mammal, or human, such as reducing and/or inhibiting the function
of the estrogen receptor. A therapeutically effective amount may be
administered in one or more administrations (e.g., the agent may be
given as a preventative treatment or therapeutically at any stage
of disease progression, before or after symptoms, and the like),
applications or dosages and is not intended to be limited to a
particular formulation, combination or administration route. It is
within the scope of the present disclosure that the SERM may be
administered at various times during the course of treatment of the
subject. The times of administration and dosages used will depend
on several factors, such as the goal of treatment (e.g., treating
v. preventing), condition of the subject, etc. and can be readily
determined by one skilled in the art.
[0069] "Estrogen dependent cancer" or "estrogen receptor positive
cancer" as used interchangeably herein refers to a tumor that
contains estrogen receptor (ER) positive cells, i.e., cells that
have estrogen receptors, and respond to the presence of estrogen
with increased proliferation. Estrogen dependent cancers may
include breast cancer, ovarian cancer, or endometrial cancer.
"Estrogen receptor positive breast cancer" is a type of breast
cancer that is sensitive to estrogen.
[0070] "Estrogen receptor" or "ER" as used interchangeably herein
refers to a receptor that is activated by the hormone estrogen and
is a member of the nuclear hormone family of intracellular
receptors. There are two different isoforms of estrogen receptor,
referred to as .alpha. (also referred to as "ERa") and .beta. (also
referred to as "ERb"). ERa and ERb genes are encoded by ESR1 and
ESR2 gene, respectively. Hormone-activated estrogen receptors form
dimers and may form homodimers or heterodimers. Both ERs are widely
expressed in different tissue types.
[0071] "Estrogen-receptor downregulators" as used herein refers to
a drug or compound which binds and down-regulates the expression of
an estrogen-receptor.
[0072] "Estrogen receptor negative breast cancer" or "Estrogen
independent breast cancer" as used interchangeably herein refers to
a tumor that does not contain estrogen receptor positive cells,
i.e., cells that lack estrogen receptors, and does not depend on
the presence of estrogen for ongoing proliferation.
[0073] "HER2 intervention drug" or "HER2 inhibitor" as used
interchangeably herein refers to a compound that targets human
Epidermal Growth Factor Receptor 2 (HER2). HER2 is a member of the
epidermal growth factor receptor family and is involved in the
development and progression of certain aggressive types of breast
cancer, such as estrogen dependent breast cancer. A HER2 inhibitor
may be a tyrosine kinase or a monoclonal antibody.
[0074] "Metastatic cancer" as used herein refers to a cancer that
has spread from the part of the body where it started (the primary
site) to other parts of the body.
[0075] "Progesterone receptor positive cancer" as used herein
refers to a tumor that contains progesterone receptor positive
(PR+) cells, i.e., cells that have progesterone receptors, which
respond to the presence of progesterone with increased
proliferation.
[0076] "Selective estrogen receptor degraders" or "SERDs" as used
interchangeably herein refers to a compound that interacts with an
ER and induce a conformational change that results in the
degradation of the receptor.
[0077] "Selective estrogen receptor modulators" or "SERMs" as used
interchangeably herein refers to a compound that interacts with an
ER and whose relative agonist/antagonist activities are manifest in
a cell selective manner. The prevention of estrogen binding to the
estrogen receptor may lead to decreased proliferation of estrogen
dependent cancer cells.
[0078] The term "subject", "patient" or "subject in the method" as
used herein interchangeably, means any vertebrate, including, but
not limited to, a mammal (e.g., cow, pig, camel, llama, horse,
goat, rabbit, sheep, hamsters, guinea pig, cat, dog, rat, and
mouse, a non-human primate (for example, a monkey, such as a
cynomolgus or rhesus monkey, chimpanzee, etc.) and a human).
[0079] In some embodiments, the subject or subject may be a human
or a non-human. In some embodiments, the subject may be a human
subject at risk for developing or already suffering from
cancer.
[0080] "Tamoxifen resistant breast cancer" as used herein refers to
a breast cancer that does respond to treatment with tamoxifen.
[0081] "Treat," "treating," or "treatment" are each used
interchangeably herein to describe reversing, alleviating, or
inhibiting the progress of a disease, or one or more symptoms of
such disease, to which such term applies. Depending on the
condition of the subject, the term also refers to preventing a
disease, and includes preventing the onset of a disease, or
preventing the symptoms associated with a disease. A treatment may
be either performed in an acute or chronic way. The term also
refers to reducing the severity of a disease or symptoms associated
with such disease prior to affliction with the disease. Such
prevention or reduction of the severity of a disease prior to
affliction refers to administration of the SERM to a subject that
is not at the time of administration afflicted with the disease.
"Preventing" also refers to preventing the recurrence of a disease
or of one or more symptoms associated with such disease.
"Treatment" and "therapeutically," refer to the act of treating, as
"treating" is defined above.
2. Methods of Treating Cancer with a SERM
[0082] The present invention is directed to methods of treating a
subject suffering from cancer. The methods include administering a
compound, i.e., SERM, having formula I:
##STR00010##
wherein [0083] TZ represents a C.sub.1-C.sub.4 alkylene group or
--CR.sup.f'R.sup.g'--CH.sub.2--O-- wherein R.sup.f' and R.sup.g'
independently represent hydrogen or a C.sub.1-C.sub.6 alkyl group;
[0084] A represents a 5- to 14-membered heteroarylene group which
may have a substituent or a C.sub.6-C.sub.14 arylene group which
may have a substituent; [0085] Y represents --CH.sub.2--NR.sup.c--
wherein R.sup.c represents hydrogen or a C.sub.1-C.sub.6 alkyl
group which may have a substituent; [0086] ring G represents the
following formula:
[0086] ##STR00011## [0087] R' represents 1 to 4 substituents
independently selected from a hydrogen atom, a C.sub.1-C.sub.6
alkoxy group, and a hydroxyl group; [0088] a partial structure in
formula (I) represented by the following formula:
[0088] ##STR00012## [0089] R'' represents hydrogen, a hydroxyl
group that may be further protected by a protecting group or a
C.sub.1-C.sub.6 alkoxy group which may have a substituent; and
[0090] R.sup.a and R.sup.b are the same as or different from each
other and each represents a hydrogen atom, a C.sub.1-C.sub.6 alkyl
group which may have a substituent, or a C.sub.3-C.sub.8 cycloalkyl
group which may have a substituent, or when R.sup.a and R.sup.b are
bonded together, they may form, together with the nitrogen atom
that is adjacent to R.sup.a and R.sup.b, a 4- to 10-membered single
ring which may have a substituent; and [0091] L represents a single
bond, or a salt thereof.
[0092] In certain embodiments, R.sup.a and R.sup.b independently
may represent a hydrogen atom, a methyl group, an ethyl group, a
n-propyl group, an iso-propyl group, a n-butyl group, an iso-butyl
group, or a tert-butyl group. -T-Z-- may represent
--CH.sub.2CH.sub.2-- or --C(CH.sub.3).sub.2CH.sub.2O--. Y may
represent --CH.sub.2--N(CH.sub.2CH.sub.3)-- or
--CH.sub.2--N(CH.sub.2CH.sub.2OH)--. Each of R'' independently may
represents a hydrogen atom or a methoxy group. R'' may represents a
hydroxyl group. In certain embodiments, the compound may be
(R)-6-{2-{ethyl[4-(2-ethylaminoethyl)benzyl]amino}-4-methoxyphenyl}-5,6,7-
,8-tetrahydronaphthalen-2-ol. Examples of other SERMS are described
in U.S. Pat. No. 7,612,114, U.S. U.S. Pat. Nos. 7,960,412,
8,399,520, U.S. Patent Publication No. US 2009-0325930, and U.S.
Patent Publication No. US 2006-0116364, the contents of which are
incorporated by reference in their entirety. An effective amount of
the compound may be administered.
(a) Dosages
[0093] In general, the dosage of administered SERM will vary
depending upon such factors as the patient's age, weight, height,
sex, general medical condition, and previous medical history.
Typically, it is desirable to provide the recipient with a dosage
of SERM, which is in the range of from about 1 pg/kg to 10 mg/kg
(amount of agent/body weight of patient), although a lower or
higher dosage also may be administered as circumstances dictate.
Dosage regimens may be adjusted to provide the optimum desired
response (e.g., a therapeutic or prophylactic response). For
example, a single bolus may be administered, several divided doses
may be administered over time or the dose may be proportionally
reduced or increased as indicated by the exigencies of the
therapeutic situation. It is especially advantageous to formulate
parenteral compositions in dosage unit form for ease of
administration and uniformity of dosage. Dosage unit form as used
herein refers to physically discrete units suited as unitary
dosages for the mammalian subjects to be tested; each unit
containing a predetermined quantity of active compound calculated
to produce the desired therapeutic effect in association with the
required pharmaceutical carrier. The specification for the dosage
unit forms of the present invention are dictated by and directly
dependent on (a) the unique characteristics of the active compound
and the particular therapeutic or prophylactic effect to be
achieved and (b) the limitations inherent in the art of compounding
such an active compound for the treatment of sensitivity in
individuals.
[0094] An exemplary, non-limiting range for a therapeutically or
prophylactically effective amount of the SERM is a dose of between
0.1 and 200 mg/kg, for example between 0.1 and 10 mg/kg, or about
20 mg/kg to about 100 mg/kg. The therapeutically or
prophylactically effective amount of the SERM may be between 1 and
200 mg/kg, 10 and 200 mg/kg, 20 and 200 mg/kg, 50 and 200 mg/kg, 75
and 200 mg/kg, 100 and 200 mg/kg, 150 and 200 mg/kg, 50 and 100
mg/kg, 5 and 10 mg/kg, or 1 and 10 mg/kg. It is to be noted that
dosage values may vary with the type and severity of the condition
to be alleviated.
[0095] In some embodiments, the SERM can be administered to a
patient in an amount of about 10 mg/day to about 500 mg/day, about
10 mg/day to about 200 mg/day (e.g., 10, 20, 30, 40, 50, 60, 70,
80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200
mg/day), 20 mg/day to about 100 mg/day, 100 mg/day to about 200
mg/day, or about 200 mg/day to about 500 mg/day (e.g., 190, 200,
210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330,
340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460,
470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590,
600, 610, 620, 630, 640, 650, 660, 670, 680, 690, or 700 mg/day),
inclusive of any single or multi-dose daily administration regimen
that falls within that total daily dose range. In some embodiments,
the dose is from about 20 mg/day to about 100 mg/day. Additionally,
one of ordinary skill in the art would also know how to adjust or
modify variables such as dosage, dosage schedules, and routes of
administration, as appropriate, for a given subject.
[0096] Further, the SERM dose may be determined by a person skilled
in the art and may vary according to factors such as the disease
state, age, sex, and weight of the individual, and the ability of
the SERM to elicit a desired response in the individual. The dose
is also one in which toxic or detrimental effects, if any, of the
SERM are outweighed by the therapeutically beneficial effects. It
is to be further understood that for any particular subject,
specific dosage regimens should be adjusted over time according to
the individual need and the professional judgment of the person
administering or supervising the administration of the
compositions, and that dosage ranges set forth herein are exemplary
only and are not intended to limit the scope or practice of the
claimed composition.
(b) Rad 1901
[0097] The SERM may be RAD 1901. Although RAD 1901 may effectively
treat vasomotor symptoms, its pharmacology is complex. Treatment of
ER positive breast cancer cells with RAD 1901 in MCF7 and BT483
cells resulted in a pronounced dose dependent down regulation of
the receptor expression. RAD 1901 is a unique SERM in that it
apparently has a relative agonist/antagonist activity in the brain
that is determined by dose. At low doses RAD 1901, behaves as a
SERM as it exhibits estrogenic activity. At high doses, RAD 1901
may function as a SERD, reversing the response.
[0098] While targeting of the estrogen signaling axis has proven
effective in the treatment of breast cancer and osteoporosis,
implementing a safe therapy that mitigates the vasomotor. While
efforts are being made to address this unmet medical need using
tissue specific estrogen complexes (TSECs) that combine estrogens
and SERMs with the intention of inhibiting estrogen action only in
some tissues (i.e., breast and uterus), preliminary clinical data
suggest that RAD 1901 may accomplish the same medical goal without
exposing the patient to estrogen. The apparent dose dependent down
regulation of ER by RAD1901 suggest that at a therapeutic (low)
dose, RAD1901 may be mediating some level of agonist activity,
while a higher dose results in more extensive SERM activity and an
effective blockade in estrogen signaling, thereby exacerbating
vasomotor symptoms.
[0099] The binding of RAD1901 enables the presentation of
protein-protein interaction surfaces within the hinge region
(flexible structure linking the DNA and hormone binding domains) of
ESR1 that are involved with transcriptional activity.
Identification of the coregulators that interact with these
surfaces on ESR1 enable a definition of their importance in RAD1901
pharmacology. As the occupancy of the receptor increases, it is
targeted for degradation, which results in a quantitative
inhibition of ESR1 signaling. This pharmacological profile
resembles that of a classical agonist, such as 17.beta.-estradiol,
where the agonist signal is terminated by proteasome-dependent
receptor degradation. The pharmacological actions of RAD1901
represent an uncoupling of ESR1-dependent transcriptional
activation from degradation. At appropriate doses, the SERD
activity manifested by RAD1901 may result in useful clinical
activity in breast cancer.
[0100] As disclosed herein, RAD1901 inhibited estrogen activation
of ESR1 in vitro and in vivo, inhibited estrogen-dependent breast
cancer cell proliferation and xenograft tumor growth, and mediated
dose-dependent downregulation of ESR1 protein. Doses of RAD1901
that were insufficient to induce ESR1 degradation were shown to
result in activation of ESR1 target genes and in stimulation of
xenograft tumor growth. RAD1901 may be used as targeted therapy for
the treatment of breast cancer brain metastases.
[0101] In some embodiments, a low dose of RAD 1901 may be about 0
mg/kg to about 25 mg/kg, about 0 mg/kg to about 20 mg/kg, about 0
mg/kg to about 15 mg/kg, about 0 mg/kg to about 10 mg/kg, about 0
mg/kg to about 5 mg/kg, about 1 mg/kg to about 25 mg/kg, about 1
mg/kg to about 20 mg/kg, about 1 mg/kg to about 15 mg/kg, about 1
mg/kg to about 10 mg/kg, about 1 mg/kg to about 5 mg/kg, about 2
mg/kg to about 25 mg/kg, about 2 mg/kg to about 20 mg/kg, about 2
mg/kg to about 15 mg/kg, about 2 mg/kg to about 10 mg/kg, about 2
mg/kg to about 5 mg/kg, about 3 mg/kg to about 25 mg/kg, about 3
mg/kg to about 20 mg/kg, about 3 mg/kg to about 15 mg/kg, about 3
mg/kg to about 10 mg/kg, about 3 mg/kg to about 5 mg/kg, about 4
mg/kg to about 25 mg/kg, about 4 mg/kg to about 20 mg/kg, about 4
mg/kg to about 15 mg/kg, about 4 mg/kg to about 10 mg/kg, about 4
mg/kg to about 5 mg/kg, about 5 mg/kg to about 25 mg/kg, about 5
mg/kg to about 20 mg/kg, about 5 mg/kg to about 15 mg/kg, about 5
mg/kg to about 10 mg/kg, about 5 mg/kg to about 7.5 mg/kg. In some
embodiments, a low dose of RAD 1901 may be less than about 25
mg/kg, about 24 mg/kg, about 23 mg/kg, about 22 mg/kg, about 21
mg/kg, about 20 mg/kg, about 19 mg/kg, about 18 mg/kg, about 17
mg/kg, about 16 mg/kg, about 15 mg/kg, about 14 mg/kg, about 13
mg/kg, about 12 mg/kg, about 11 mg/kg, about 10 mg/kg, about 9
mg/kg, about 8 mg/kg, about 7 mg/kg, about 6 mg/kg, about 5 mg/kg,
about 4 mg/kg, about 3 mg/kg, about 2 mg/kg, or about 1 mg/kg.
[0102] In some embodiments, a high dose of RAD 1901 may be about 15
mg/kg to about 500 mg/kg, about 15 mg/kg to about 250 mg/kg, about
15 mg/kg to about 200 mg/kg, about 15 mg/kg to about 150 mg/kg,
about 15 mg/kg to about 100 mg/kg, about 15 mg/kg to about 75
mg/kg, about 20 mg/kg to about 500 mg/kg, about 20 mg/kg to about
250 mg/kg, about 20 mg/kg to about 200 mg/kg, about 20 mg/kg to
about 150 mg/kg, about 20 mg/kg to about 100 mg/kg, about 20 mg/kg
to about 75 mg/kg, about 25 mg/kg to about 500 mg/kg, about 25
mg/kg to about 250 mg/kg, about 25 mg/kg to about 200 mg/kg, about
25 mg/kg to about 150 mg/kg, about 25 mg/kg to about 100 mg/kg, or
about 25 mg/kg to about 75 mg/kg. In some embodiments, a high dose
of RAD 1901 may be more than about 15 mg/kg, 20 mg/kg, about 25
mg/kg, about 30 mg/kg, about 35 mg/kg, about 40 mg/kg, about 45
mg/kg, about 50 mg/kg, about 55 mg/kg, about 60 mg/kg, about 65
mg/kg, about 70 mg/kg, about 75 mg/kg, about 80 mg/kg, about 85
mg/kg, about 90 mg/kg, about 95 mg/kg, about 100 mg/kg, about 105
mg/kg, about 110 mg/kg, about 115 mg/kg, about 120 mg/kg, about 125
mg/kg, about 130 mg/kg, about 135 mg/kg, about 140 mg/kg, about 145
mg/kg, about 150 mg/kg, about 155 mg/kg, about 160 mg/kg, about 165
mg/kg, about 170 mg/kg, about 175 mg/kg, about 180 mg/kg, about 185
mg/kg, about 190 mg/kg, about 195 mg/kg, about 200 mg/kg, about 250
mg/kg, about 300 mg/kg, about 350 mg/kg, about 400 mg/kg, about 450
mg/kg or about 500 mg/kg.
[0103] In some embodiments, RAD1901 can be administered to a
patient in an amount of about 10 mg/day to about 500 mg/day, about
10 mg/day to about 200 mg/day (e.g., 10, 20, 30, 40, 50, 60, 70,
80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200
mg/day), 20 mg/day to about 100 mg/day, 100 mg/day to about 200
mg/day, or about 200 mg/day to about 500 mg/day (e.g., 190, 200,
210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330,
340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460,
470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590,
600, 610, 620, 630, 640, 650, 660, 670, 680, 690, or 700 mg/day),
inclusive of any single or multi-dose daily administration regimen
that falls within that total daily dose range. In some embodiments,
the dose is from about 20 mg/day to about 100 mg/day. Additionally,
one of ordinary skill in the art would also know how to adjust or
modify variables such as dosage, dosage schedules, and routes of
administration, as appropriate, for a given subject.
3. Methods of Treating Estrogen Receptor Positive Cancer of the
Brain
[0104] The methods described above may be used to treat an estrogen
receptor positive cancer of the brain. In some embodiments, the
cancer may include subtypes of brain tumors that may express ER,
such as Breast Cancer Brain Metastases (BCBM), Astrocytoma,
Chondrosarcoma, Craniopharyngioma, Glioblastoma, Glioma,
Hemangioma, Medulloblastoma, Meningioma, Neurofibroma, Neuronal and
Mixed Neuronal-Glial Tumors, Oligoastrocytoma, Pituitary Tumor,
PNET--(primitive neuroectodermal tumor), Schwannomak, or
Leptomeningeal metastases. In some embodiments, the cancer may be
other cancers such as Atypical Teratoid Rhabdoid Tumor (ATRT),
Choroid Plexus Carcinoma, Ependymoma, Germ Cell Tumor, Juvenile
Pilocytic Astrocytoma, Oligodendroglioma, or Pineal Tumor.
(a) Breast Cancer Brain Metastases
[0105] The methods described above may be used to treat a subject
suffering from breast cancer brain metastases. 10-20% of breast
cancer patients ultimately experience breast cancer metastasis to
the brain, i.e., BCBM. 30-40% of BCBM express ER. ER expression is
retained in 50-65% of BCBM that arise from ER+/PR+ tumors, despite
treatment of the initial tumor with endocrine therapies. As many as
50% of BCBM express the estrogen receptor, and the brain is an
environment rich in aromatase activity, suggesting that estrogen
levels and signaling may be of importance in the establishment and
maintenance of BCBM. Therefore, the availability of the SERM, such
as RAD 1901 or a similar SERM with significant brain penetrance,
that can induce estrogen receptor turnover in BCBM may provide
therapeutic benefit in the treatment of BCBM.
[0106] Few treatments can access the brain, one of three most
frequent sites of metastasis for breast cancer, leaving targeted or
whole brain radiation as the standard of care for patients
diagnosed by BCBM. In the majority of BCBM occurring in patients
diagnosed with ESR1-positive breast tumors, ESR1 expression is
retained, and the high expression of aromatase in the brain
suggests that the disruption of ESR1 signaling may be beneficial in
this setting. Therefore, the availability of the SERM, such as
RAD1901 or a similar SERM with significant brain penetrance, that
can induce estrogen receptor turnover in BCBM may provide
therapeutic benefit in the treatment of BCBM.
(b) Astrocytoma
[0107] Astrocytoma is a type of cancer of the brain that originate
in astrocytes, which are a particular kind of glial cells,
start-shaped brains cells in the cerebrum. Low ERb expression has
been shown to be associated with the progression of
astrocytoma.
(c) Chondrosarcoma
[0108] Chondrosarcoma is a type of tumor that affects the bones and
joints. Chondrosarcoma grow from the types of cells that make
cartilage in the skull. In the head, these tumors grow inside the
bones at the base of the back part of the skull and may be very
close to the nerves and blood vessels around the brainstem. ER is
present and active in chondrosarcoma tumors.
(d) Craniopharyngioma
[0109] Craniopharyngioma is a benign tumor that develops near the
pituitary gland. ER may be present in craniopharyngioma and may be
associated with improved disease prognosis.
(e) Glioblastoma multiforme
[0110] Glioblastoma multiforme, also known as "glioblastoma," is
the most common and most aggressive malignant primary brain tumor
in humans, involving glial cells and accounting for 52% of all
functional tissue brain tumor cases and 20% of all intracranial
tumors. Glioblastoma may express both ERs, which may play a role in
etiology and treatment.
(f) Glioma
[0111] Glioma is a type of tumor that starts in the brain or spine
and arises from glial cells. Gliomas make up approximately 30% of
all brain and central nervous system tumors and 80% of all
malignant brain tumors. Glioma may express both ERs, which may play
a role in etiology and treatment. Glioma may be responsive to
tamoxifen treatment.
(g) Hemangioma
[0112] Hemangioma is a benign and usually self-involuting tumor
(swelling or growth) of the endothelial cells that line blood
vessels. Hemangioma is characterized by increased number of normal
or abnormal vessels filled with blood. Human vascular endothelial
cells express ER isoforms and are responsive to tamoxifen
treatment. Hemangioma may be intracranial hemangiomas or cutaneous
hemangiomas.
(h) Medulloblastoma
[0113] Medulloblastoma is a highly malignant primary brain tumor
that originates in the cerebellum or posterior fossa.
Medulloblastomas may originate from immature or embryonal cells at
their earliest stage of development. Medulloblastoma may express ER
isoforms. ER isoforms are associated with growth and migration of
these cells.
(i) Meningioma
[0114] Meningiomas are a diverse set of tumors arising from the
meninges, i.e., the membranous layers surrounding the central
nervous system. Meningioma may express both ER isoforms. Meningioma
may be responsive to tamoxifen treatment.
j) Neurofibroma
[0115] Neurofibroma is a benign nerve sheath tumor in the
peripheral nervous system.
[0116] Neurofibromas arise from nonmyelinating-type Schwann cells
that exhibit biallelic inactivation of the NF1 gene that codes for
the protein neurofibromin. Neurofibroma may be ER positive.
(k) Neuronal & Mixed Neuronal-Glial Tumors
[0117] Neuronal & Mixed Neuronal-Glial Tumors are rare, benign
tumors that come from ganglion-type cells, i.e., groups of nerve
cells. ER may be present in these tumors.
(l) Oligoastrocytoma
[0118] Oligoastrocytomas are a subset of brain tumors that present
with an appearance of mixed glial cell origin, astrocytoma and
oligodendroglioma. Oligoastrocytoma may have a lasting response to
tamoxifen treatment.
(m) Pituitary Tumor
[0119] A pituitary tumor is an abnormal growth in the pituitary
gland. Both ER.alpha. and ERb may be detected in pituitary
tumors.
(n) Primitive Neuroestodermal Tumor (PNET)
[0120] Primitive neuroestodermal tumor is a neural crest tumor. The
majority of the cells in the PNET are derived from neuroectoderm
but have not developed and differentiated in the way a normal
neuron would, and so the cells appear "primitive. ERa may be
present and may increase metastatic potential via extracellular
signal-regulated Kinase (ERK) activation.
(o) Schwannoma
[0121] Schwannoma (also known as an "neurilemmoma," "neurinoma,"
"neurolemmoma," and "Schwann cell tumor") is a benign nerve sheath
tumor composed of Schwann cells, which normally produce the
insulating myelin sheath covering peripheral nerves. Schwannoma may
express ER.alpha..
(p) Leptomeningeal Metastases
[0122] Leptomeningeal metastasis is breast cancer metastasis to the
membranes (meninges) surrounding the brain and spinal cord. A
durable response has been observed in patient(s) treated with
aromatase inhibitors, which suggests possible responsiveness to ER
targeted therapies.
4. Methods of Treating a Cancer that is Resistant to an Estrogen
Receptor Modulator
[0123] The methods described above may be used to treat a cancer
that is resistant to an estrogen receptor modulator. The resistance
to the estrogen receptor modulator may be acquired. The estrogen
receptor modulator may be a selective estrogen receptor modulator
(SERM). The SERM may be tamoxifen, idoxifene, raloxifene, or ICI
182,780. The cancer may be breast, endometrial or ovarian cancer.
The cancer may be tamoxifen resistant breast cancer.
[0124] Because patients diagnosed with BCBM are likely to have
already progressed following endocrine therapies, RAD1901 may be
used in breast tumors resistant to tamoxifen and/or aromatase
inhibitors. RAD1901 may be beneficial in the BCBM setting when
combined with additional therapeutic(s) that will ensure the
inhibition of growth of peripheral metastases.
5. Mechanisms of Delivery
[0125] The SERM may be formulated to be compatible with its
intended route of administration. Examples of routes of
administration include, but are not limited to, parenteral, e.g.,
intravenous, intradermal, subcutaneous, oral, intranasal (e.g.,
inhalation), transdermal (e.g., topical), transmucosal, and rectal
administration. In a specific embodiment, the SERM is formulated in
accordance with routine procedures as a pharmaceutical composition
adapted for intravenous, subcutaneous, intramuscular, oral,
intranasal, or topical administration to human beings. Typically,
compositions for intravenous administration are solutions in
sterile isotonic aqueous buffer. Where necessary, the composition
may also include a solubilizing agent and a local anesthetic such
as lignocaine to ease pain at the site of the injection.
[0126] Various delivery systems are known and can be used to
administer one or more SERMs or the combination of one or more
SERMs and a prophylactic agent or therapeutic agent useful for
preventing, managing, treating, or ameliorating a disorder or one
or more symptoms thereof, e.g., encapsulation in liposomes,
microparticles, microcapsules, receptor-mediated endocytosis (see,
e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987)), etc. Methods
of administering a prophylactic or therapeutic agent of the SERM
include, but are not limited to, parenteral administration (e.g.,
intradermal, intramuscular, intraperitoneal, intravenous and
subcutaneous), epidural administration, intratumoral
administration, and mucosal administration (e.g., intranasal and
oral routes).
6. Combination Treatments
[0127] The methods described above may include a combination
treatment of the compound of formula I with other drugs and/or
other conventional cancer therapies, such as hormone therapy.
(a) Combination Drugs
[0128] The methods may further include administering an effective
amount of at least one compound of a cyclin-dependent kinase 4 and
6 inhibitor (CDK4/6 inhibitor), an antiestrogen, a ligand of
retinoic acid or retinoic X receptor, an antiprogestin, an
antiandrogen, vitamin D or metabolite thereof, a farnesyl
transferase inhibitor, a PPAR.alpha. or gamma agonist and a MAP
kinase inhibitor.
(b) Conventional Cancer Therapies
[0129] Conventional cancer therapies may include surgery, radiation
therapy, chemotherapy, hormone therapy, and targeted therapy.
Examples of surgery include open craniotomy with maximal excision,
which may be followed by radiation therapy. Examples of radiation
therapy include whole-brain irradiation, fractionated radiotherapy,
and radiosurgery, such as stereotactic radiosurgery, e.g., Gamma
Knife radiosurgery. Examples of chemotherapy include
anthracyclines, such as doxorubicin (Adriamycin, Doxil), epirubicin
(Ellence), and daunorubicin (Cerubidine, DaunoXome), capecitabine
(Xeloda), carboplatin (Paraplatin), cisplatin, cyclophosphamide
(Cytoxan), eribulin (Halaven), fluorouracil (also called
5-fluorouracil or 5-FU; Adrucil), gemcitabine (Gemzar), ixabepilone
(Ixempra), methotrexate (Amethopterin, Mexate, Folex), mitoxantrone
(Novantrone), mutamycin (Mitomycin), taxanes, such as paclitaxel
(Taxol, Abraxane), and docetaxel (Taxotere), thiotepa (Thioplex),
vincristine (Oncovin, Vincasar PES, Vincrex), and vinorelbine
(Navelbine). Examples of targeted therapy include trastuzumab
(Herceptin), lapatinib (Tykerb), bevacizumab (Avastin), pertuzumab
(Perjeta), and everolimus (Afinitor).
[0130] i. Endocrine Therapy (Hormone Therapy)
[0131] Endocrine therapy, also known as hormonal therapy, hormone
therapy, and hormone treatment, is a treatment that adds, blocks,
or removes hormones. For example, hormones may be given to adjust
low hormone levels. Synthetic hormones or other drugs may be given
to block the body's natural hormones to slow or stop the growth of
certain cancers (such as prostate and breast cancer). Endocrine
therapy may also include surgery to remove the gland that makes a
certain hormones.
[0132] Examples of hormone therapy include selective estrogen
receptor modulators (SERMs), such as tamoxifen, raloxifene,
endoxifene, toremifene, lasofoxifene, pipendoxifene, bazedoxifene,
and ospemifene, aromatase inhibitors, such anastrozole, letrozole,
exemestane, formestane, fadrozole, aminoglutethimide, and
testolactone, a HER2 intervention drug, such as a HER2 inhibitor,
such as Herceptin (trastuzumab), pertuzumab, and lapatinib, and
estrogen-receptor downregulators, such as fulvestrant (ICI
182,780).
7. Subject or Subject in the Method
[0133] The methods described above are directed to treating a
subject with a SERM. The subject treated by the methods described
above may be a subject or patient suffering from or at risk of
suffering from an estrogen receptor positive cancer of the brain,
such as BCBM, or a cancer that is resistant to an estrogen receptor
modulator, such as tamoxifen resistant breast cancer. The subject
may be diagnosed or identified as having or at risk of having
cancer using known methods and assays, such as a biopsy. The
subject may be treated with SERM alone or in combination with
another drug and/or conventional cancer therapy, as described
above. The subject may be treated with the SERM as a neoadjuvant
therapy or post-surgery. The present invention has multiple
aspects, illustrated by the following non-limiting examples.
EXAMPLES
[0134] The foregoing may be better understood by reference to the
following examples, which are presented for purposes of
illustration and are not intended to limit the scope of the
invention.
Example 1
In Vitro Analysis of ER Degradation
[0135] 48 hours prior to treatment, MCF7 cells were plated in
phenol red free DMEM/F12 media supplemented with 8% charcoal
stripped fetal bovine serum, non-essential amino acids, and sodium
pyruvate. After 20 hours of treatment with the indicated ligands,
i.e., estradiol ("E2"; Sigma-Aldrich), antiestrogen ICI 182,780
("IC"; Sigma-Aldrich), and 4-hydroxytamoxifen ("40HT";
Sigma-Aldrich), (0-1 .mu.M), cells were washed and lysed in RIPA
lysis buffer (50 mM Tris, pH 8, 150 mM NaCl, 1% IGEPAL, 0.02% SDS,
0.5% sodium deoxycholate, 1 mM EDTA). 50 .mu.g of cleared lysate
was resolved by SDS-PAGE and analyzed by immunoblot detection of
ER.alpha. or cytokeratin 18 (loading control), as illustrated in
FIG. 1A.
Example 2
In Vivo Analysis of RAD1901 in MCF7 Xenograft Tumors
[0136] A total of 90 female Nu/Nu mice were ovariectomized and
implanted subcutaneous simultaneously with an estrogen pellet
(Innovative Research of America) releasing 0.72 mg estradiol (E2)
over 60 days. 2 days later an approximately 6 mm3 fragment of an
MCF7 xenograft tumor (isolated from a recently sacrificed estrogen
treated nu/nu mouse) was inserted subcutaneous into the axial
mammary gland. Tumor growth (by caliper measurement) and animal
body weight were monitored 3.times. weekly until tumor volume
reached .about.0.2 cm.sup.3. Mice (n.about.10) were randomized to
the following groups: estrogen control (corn oil vehicle), E2+
RAD1901 (20 mg/kg; Radius Health, Inc.), E2+ Tamoxifen (20 mg/kg;
Sigma-Aldrich). Treatments were formulated in sterile corn oil and
were administered daily by subcutaneous injection. After 3 weeks of
treatments, animals were euthanized and serum and tissues saved for
analysis. FIG. 1B depicts tumor volume analyzed using non-linear
curve fit and exponential growth calculation (Graphpad Prism),
followed by two-way ANOVA and Bonferroni analysis.
Example 3
[0137] Mechanism by which RAD1901 Downregulates ER Expression
[0138] While the reduced levels of ER following treatment with
RAD1901 results in receptor degradation, whether the drug
influences the transcriptional activity of the gene encoding ER was
determined (FIG. 2). MCF7 cells were pre-treated with Vehicle (Veh)
or translation (cyclohexamide--CHX, 10 .mu.g/ml), proteasome
(MG132, 30 .mu.M) or transcription (Actinomycin D--Actin. D, 100
ng/ml) inhibitors for 2 hours prior to 6 hours of treatment with
RAD1901 (0.1 or 1 .mu.M), or 0.1 .mu.M ICI 182,780 (ICI) or
raloxifene (Ral). FIG. 2A shows ER.alpha. protein expression in
whole cell extracts was analyzed by immunoblot, as in Example 1.
FIG. 2B shows cells treated, as indicated, were washed in PBS prior
to lysis. RNA isolation (BioRad) and reverse transcription
(iScript; BioRad) were performed per kit manufacturer's
instructions. qRT-PCR of cDNA was done using iQ SYBR Green Supermix
(Bio-Rad) per kit instructions and performed using the iCycler
optical system with associated software (Bio-Rad). mRNA abundance
was calculated using the .DELTA..DELTA.CT method to normalize
ER.alpha. mRNA expression to similarly detected housekeeping gene
36B4.
Example 4
Conformational Changes Induced in ER as a Consequence of Binding of
RAD1901
[0139] A series of short peptides whose ability to interact with
ER.alpha. is influenced by the nature of the bound ligand were
previously identified (see Chang et al. Methods Enzymol. (2003)
364: 118-42; Huang et al. Mol Endocrinol. (2002) 16(8): 1778-92;
Connor et al. Cancer Res. (2001) 61(7): 2917-22; Chang et al. Mol
Cell Biol. (1999) 19(12):8226-39; and Norris et al. Science (1999)
285(5428): 744-6). The interaction of these "conformational probes"
can be measured in vitro or within intact cells and thus enables
the definition of ligand induced conformational changes in the
receptor. This is significant, since differences in receptor
conformation facilitate the engagement of different coregulatory
proteins resulting in different pharmacological activities.
Application of this technology has led to the determination that
their impact on receptor structure is a distinguishing feature of
ER ligands.
[0140] FIG. 3 shows that RAD1901 induces a unique conformation of
ER.alpha. as shown in the interaction between ER and
conformation-specific peptides in a mammalian two-hybrid system.
Triplicate wells of SKBR3 cells were transfected (Lipofectin per
manufacturer's instructions) with plasmids expressing ER.alpha.
fused to VP16 together with Gal4DBD alone (control) or fused to ER
interacting peptides noted on the horizontal axis. Cells were then
treated with RAD1901 (1 nM-1 .mu.M) or the indicated ER ligands
(100 nM). Interaction of ER.alpha. with the Gal4DBD peptide
constructs was detected through activation of a Gal4 responsive
luciferase reporter construct and was normalized to detected
.beta.-galactosidase activity generated by a co-transfected
constitutive expression vector. Ligand classes recognized by each
probe are indicated below the graph.
Example 5
[0141] RAD1901 Possesses Dose Dependent Agonist and/or Antagonist
Potential
[0142] The complex pharmacological activities exhibited by RAD1901
suggest that it will exhibit a unique gene expression profile in
target cells. A recent extensive microarray analysis has identified
"sentinel" subsets of ER-responsive genes that can differentiate
between ER agonists, SERMs and SERDs. For example, some of these
are responsive to only the SERM tamoxifen, while others display a
graded response to SERMs with varying agonist/antagonist potential,
and yet others are repressed by agonists or SERMs and are induced
only by SERDs. RAD1901 exhibits dose dependent agonist/antagonist
regulation of ER transactivation of target genes (FIG. 4). MCF7
breast cancer cells were treated 24 hours with RAD1901 (0-1 .mu.M)
in the presence or absence of 17.beta.-estradiol (1 nM). RNA
isolation and the analysis of the expression of target genes
Anterior gradient protein 2 (AGR2) and KCNK6 was conducted as in
Example 3.
Example 6
Materials and Methods
[0143] Reagents. Purchased ESR1 ligands included 17.beta.-estradiol
(Sigma), ICI 182,780 (Tocris), tamoxifen (Sigma), raloxifene
(Tocris), and 4-hydroxytamoxifen (Sigma).
(R)-6-(2-(N-(4-(2-(ethylamino)ethyl)benzyl)-N-ethylamino)-4-methoxyphenyl-
)-5,6,7,8-tetrahydronaphthalen-2-ol dihydrochloride was provided by
Radius Pharmaceuticals. Bazedoxifene and GW7604 were synthesized as
previously described (Miller et al., (2001) J Med Chem 44
(11):1654-1657; U.S. Pat. No. 5,681,835). Ligands were dissolved in
ethanol or DMSO.
[0144] Cell culture. MCF7 and SKBR3 cell lines were maintained in
DMEM/F12 or RPMI media (Invitrogen), respectively, supplemented
with 8% fetal bovine serum (FBS, Gemini), non-essential amino acids
(Invitrogen), and sodium pyruvate (Invitrogen). Unless otherwise
indicated, cells were plated for experiments in media lacking
phenol red and supplemented with 8% charcoal stripped FBS (CFS,
Gemini). LTED MCF7 cells were maintained and plated for experiments
in phenol red free DMEM/F12 media supplemented with 8% CFS that had
been charcoal stripped twice. 48 hours after plating, cells were
treated with ESR1 ligands as indicated, and were harvested for
immunoblot or real time quantitative PCR analysis 24 hours after
treatment. Cell lines were authenticated by STR analysis performed
by ATCC in 2013.
[0145] Immunoblot analysis. Protein expression was analyzed as
described (Wittmann et al., (2007) Cancer Res 67 (19):9549-9560)
using antibodies purchased from Sigma-Aldrich--A5441 (.beta.-actin)
and Santa Cruz Biotechnology--sc-6259 (cytokeratin 18), sc-20680
(lamin A), sc-5546 (.alpha.-tubulin) and sc-8005 (ESR1).
[0146] RNA isolation and real time quantitative PCR. RNA isolation
and analysis was performed as described (Wardell et al., (2001)
Biochem Pharmacol 82 (2):122-130). mRNA abundance was calculated
using the .DELTA..DELTA.CT method (Wardell et al., (2001) Biochem
Pharmacol 82 (2):122-130). Primer sequences are available upon
request.
[0147] Proliferation assays. Assays evaluating the effects of SERDs
and SERMs on cell proliferation were performed as described
(Wardell et al., (2013) Clin Cancer Res 19 (9):2420-2431).
[0148] Transfections. Mammalian 2-hybrid analysis of VP16-ESR1 with
conformation-selective peptide probes was performed essentially as
previously described (Wardell et al., (2012) Mol Endocrinol 26
(7):1235-1248).
[0149] In vivo studies. All applicable international, national,
and/or institutional guidelines for the care and use of animals
were followed. All procedures performed in studies involving
animals were in accordance with the ethical standards of the Duke
University Institutional Animal Care and Use Committee.
[0150] Uterine wet weight analysis: Ovariectomized (10 days prior)
female C57Bl/6 mice (Charles River) were treated daily (n=5) for 3
days with vehicle or estradiol benzoate (10 .mu.g/kg sc) as well as
vehicle, raloxifene (10 mg/kg sc), or RAD1901 (0.1-100 mg/kg sc).
Ligands were dissolved in corn oil (Spectrum chemicals). On day 4,
mice were euthanized, and tissues were retained for analysis.
Uterine wet weight was calculated as a ratio of uterus weight upon
removal to body weight post-mortem.
[0151] Xenograft tumor analysis: Estrogen-stimulated MCF7 tumors
were initiated in the axial mammary gland of 6-week old
estrogen-treated (0.72 mg/60 days pellet sc, Innovative Research of
America) ovariectomized female NU/NU mice (in-house colony) by
serial transfer and were measured as described (Wardell et al.,
(2013) Clin Cancer Res 19 (9):2420-2431). FIG. 5: At .about.0.1
cm.sup.3 tumor volume, mice were randomized (n=9-10) to daily
treatment with vehicle, RAD1901 (20 mg/kg), or tamoxifen (20
mg/kg). FIG. 9: At .about.0.1 cm.sup.3 tumor volume, the estrogen
pellet was surgically removed, and mice were randomized (n=6-10) to
daily treatment with vehicle or RAD1901 (0.3-10 mg/kg). Treatments
were formulated as above. Animal tissues were processed and
analyzed as described (Wardell et al., (2013) Clin Cancer Res 19
(9):2420-2431).
Example 7
RAD1901 Inhibits ESR1 Activity In Vitro and In Vivo
[0152] The ability of RAD1901 (FIG. 5A) to modulate the 17-.beta.
estradiol (E2) dependent transcriptional activity of the human
estrogen receptor alpha (ESR1) and human estrogen receptor beta
(ESR2) was assessed in transiently transfected SKBR3 cells using a
synthetic reporter gene. FIG. 5A shows the chemical structure of
the SERM RAD1901. For FIG. 5B, SKBR3 cells were plated in phenol
red free media supplemented with charcoal stripped FBS (CFS) 24
hours prior to transfection with an ERE-luciferase reporter
together with ESR1 or ESR2 expression vectors. 24 hours after
transfection, cells were treated with E2 (10 nM) together with
RAD1901 (10.sup.-10-10.sup.-6 M) for 24 hours prior to harvest and
analysis of luciferase activity normalized to co-transfected
.beta.-galactosidase control. For FIG. 5C, MCF7 cells were plated
in phenol red free media supplemented with CFS 48 hours prior to
treatment with 10.sup.-9 M E2 together with ICI 182,780 (ICI),
RAD1901 (RAD), GW7604, or 4-hydroxytamoxifen (40HT)
(10.sup.-11-10.sup.-6 M) for 24 hours. mRNA levels of ESR1 target
gene trefoil factor 1 (TFF1) were assessed using RT qPCR following
RNA isolation. mRNA expression was normalized to the similarly
detected 36B4 housekeeping gene, and expression levels are
presented as fold change as compared to the vehicle-treated
control. For FIG. 5D, MCF7 cells were plated in phenol red free
media supplemented with CFS 24 hours prior to treatment, and were
treated with 10.sup.-9 M E2 as well as with the indicated ligands
(10.sup.-11-10.sup.-6 M) on days 1, 4, and 6 of an 8 day
proliferation assay. DNA content as assessed by fluorescence was
measured as a surrogate for cell proliferation. The relative
increase in DNA fluorescence was calculated by normalizing to
baseline values detected in a duplicate plate of cells that was
harvested on day 1 prior to the initial treatment. Data are
representative of at least 3 independent experiments. For FIGS.
5E-5F, MCF7 cell derived tumors were implanted into ovariectomized
estrogen-treated nu/nu mice. When tumor volume reached .about.0.1
cm.sup.3, animals (n=9-10) were randomized to receive daily
treatment with vehicle, tamoxifen (Tam, 20 mg/kg sc) or RAD1901
(RAD) (20 mg/kg sc). For FIG. 5E, mean tumor volume.+-.SEM per day
of treatment was presented. Significance (2-way ANOVA of matched
values followed by Bonferroni comparison) as compared to the
vehicle control was indicate d (*p<0.0001). For FIG. 5F,
expression of ESR1 target genes in tumors was analyzed essentially
as in (C).
[0153] In this analysis it was determined that although RAD1901
effectively inhibited E2-dependent activation of an ERE-luciferase
reporter by either isoform, it was a more potent inhibitor of ESR1
(100-fold) (FIG. 5B). Similarly, it was demonstrated in MCF7 breast
cancer cells that RAD1901 inhibited E2-dependent (a) induction of
target gene transcription and (b) stimulation of cell proliferation
with an efficacy and potency similar to that of the SERMs
4-hydroxytamoxifen (40HT) and raloxifene (Ralox) and the SERDs ICI
182,780 (ICI, fulvestrant) and GW7604 (FIG. 5C-5D). To assess the
activity of RAD1901 in vivo, a xenograft tumor study was conducted
in estrogen-treated immunocompromised mice using the
well-characterized ESR1-dependent MCF7 cell model. In this study,
it was observed that RAD1901 (20 mg/kg) inhibited E2-stimulated
growth of the tumors with efficacy similar to tamoxifen (20 mg/kg).
RAD1901 and tamoxifen were also shown to suppress the expression of
the ESR1-target genes PGR and FHL1 to the same degree in treated
tumors, a result that confirms target engagement (FIGS. 5E-5F).
Example 8
RAD1901 Exhibits the Pharmacological Properties of a Selective
Estrogen Receptor Degrader (SERD)
[0154] RAD1901 not only functioned as an ESR1 antagonist, but also
downregulated the expression of this receptor. ESR1 expression was
significantly downregulated in MCF-7 cells treated with RAD1901.
For FIG. 6A, MCF7 cells were treated for 24 hours with ICI
(10.sup.-13-10.sup.-7 M) or RAD1901 (RAD), (10.sup.-11-10.sup.-5
M). Expression of ESR1 and loading control cytokeratin 18
(CK18--FIG. 7A) in whole cell extracts were detected by immunoblot
(top). ESR1 levels relative to CK18 were quantitated by
densitometry using Adobe Photoshop (bottom). For FIG. 6B, MCF7
cells were plated as in FIG. 5B prior to 1 hour pre-treatment with
vehicle or MG132 (10 .mu.g/ml), followed by 6 hours of treatment
with 10.sup.-7 M vehicle, ICI, Ral or RAD1901 (RAD),
(10.sup.-8-10.sup.-6). ESR1 expression was detected as in FIG. 6A.
For FIGS. 6C-6D, LTED MCF7 cells were plated in phenol red free
media supplemented with FBS that was stripped of growth factors
twice using charcoal. For FIG. 6C, after 48 hours, cells were
treated for 24 hours with E2 (10.sup.-7 M) or SERDs (10.sup.-6 M)
and ESR1 was analyzed as in FIG. 6A. For FIG. 6D, LTED MCF7 cells
were treated with ICI or RAD1901 (RAD), (10.sup.-11-10.sup.-6 M) on
days 1, 4, and 6 of an 8 day proliferation assay and analyzed as in
FIG. 5.
[0155] For FIG. 7A, cytokeratin 18 (CK18) loading control was
detected for samples illustrated in FIG. 6A. For FIG. 7B, BT483
cells were plated in phenol red free media supplemented with CFS 48
hours prior to treatment with 10.sup.-9 M E2 together with ICI
182,780 (ICI), RAD1901 (RAD), GW7604, or 4-hydroxytamoxifen (40HT)
(10.sup.-11-10.sup.-6 M) for 24 hours. mRNA levels of ESR1 target
gene trefoil factor 1 (TFF1) were assessed using RT qPCR following
RNA isolation. mRNA expression was normalized to the similarly
detected 36B4 housekeeping gene, and expression levels are
presented as fold change as compared to the vehicle-treated
control. For FIG. 7C, BT483 cells were treated for 24 hours with
ICI (10.sup.-13-10.sup.-7 M) or RAD1901 (RAD),
(10.sup.-11-10.sup.-5 M). Expression of ESR1 and loading control
cytokeratin 18 in whole cell extracts were detected by immunoblot
(right). ESR1 levels relative to CK18 were quantitated by
densitometry using Adobe Photoshop (left). For FIG. 7D, BT483 cells
were plated in phenol red free media supplemented with CFS 24 hours
prior to treatment, and were treated with 10.sup.-9 M E2 as well as
with the indicated ligands (10.sup.-11-10.sup.-6 M) on days 1, 4,
and 6 of an 8 day proliferation assay. DNA content as assessed by
fluorescence was measured as a surrogate for cell proliferation.
The relative increase in DNA fluorescence was calculated by
normalizing to baseline values detected in a duplicate plate of
cells that was harvested on day 1 prior to the initial treatment.
Data are representative of at least 3 independent experiments.
[0156] The downregulation of the ESR1 by RAD1901 was a significant
contributor to its antagonist efficacy (FIG. 6A). RAD1901 had no
effect on ESR1 mRNA expression (not shown). However, as observed in
ICI-treated cells, the downregulation of ESR1 by RAD1901 was
completely blocked by pre-treatment of cells with the proteasome
inhibitor MG132 (FIG. 6B). Similar results were obtained in the
BT483 breast cancer cell line (FIGS. 7B-7D). For comparative
purposes, ESR1 expression levels were evaluated in cells treated
with tamoxifen, which stabilizes ESR1 expression, and SERDs
bazedoxifene (BZA), GW7604 and ICI. These studies revealed that the
degree of downregulation of ESR1 by RAD1901 was similar to that
achieved by BZA under the same conditions (FIG. 6C).
[0157] To assess the potential significance of this SERD activity,
the activity of RAD1901 was evaluated in LTED MCF7 cells, an
accepted model of aromatase resistance in which compounds with
ER-antagonist activity alone, like tamoxifen, were minimally
effective (not shown). In this assay, it was demonstrated that,
like ICI and BZA, RAD1901 downregulated ESR1 expression and
inhibited cell proliferation (FIG. 6D). Thus, considering its
antagonist activity in several models of breast cancer and its
ability to downregulate ESR1, it was appropriate to classify
RAD1901 as a SERD.
Example 9
RAD1901 Exhibits Dose Dependent SERD Activity In Vivo
[0158] The observation that RAD1901 exhibited the pharmacological
properties of a SERD was unexpected given that it was (a)
identified in screens for compounds that manifest ESR1 agonist
activity in the CNS and (b) evaluated in clinical trials as a
potential treatment for the vasomotor symptoms (hot flashes)
associated with menopause, an indication for which only estrogens
have proven effective. However, the results of the clinical trials
for hot flashes revealed that RAD1901 exhibited a complex
pharmacology. At the lowest dose tested it appeared to effectively
suppress hot flashes but was ineffective at the higher doses
tested. This inverted U-shaped pharmacology suggested that at low
doses this compound may have favorable agonist activity, but at
higher doses the ability of RAD1901 to induce ESR1 turnover
dominates. A series of studies was performed to explore the
functional consequences of the complex pharmacological activities
of RAD1901 in vivo.
[0159] As a first step, the impact of RAD1901 on uterine wet weight
in mice was evaluated. For this study, increasing doses of RAD1901
was administered daily to ovariectomized C57Bl/6 mice receiving
vehicle alone or E2 (10 .mu.g/kg; a physiological replacement
dose). A group of mice receiving 10 mg/kg of raloxifene was
included for comparative purposes and for reference. Ovariectomized
C57Bl/6 mice (n=5) were treated daily for 3 days with (FIG. 8A)
vehicle or (FIG. 8B) estradiol (10 .mu.g/kg) together with vehicle,
Ralox (10 mg/kg) or RAD1901 (RAD), (0.3-100 mg/kg). At euthanasia,
body weight as well as uterine wet weight was measured prior to
cryopreservation of the uterus. When administered as a single
agent, a statistically significant increase in uterine weight was
observed in those animals receiving the lowest dose of RAD1901 (0.3
mg/kg). For FIG. 8C, ESR1 and .beta.-actin expression in extracts
made from pulverized uterine tissues were analyzed by immunoblot as
in FIG. 5 (left). ESR1 expression relative to .beta.-actin was
quantitated as in FIG. 6 (right). Significant downregulation
(*p<0.05) of ESR1 was determined by ANOVA followed by Bonferroni
comparison.
[0160] At doses of 1 mg/kg and above, the uterine wet weights of
treated animals were indistinguishable from vehicle treated animals
(FIG. 8A). Further, RAD1901 administration was shown to inhibit
E2-dependent increases in uterine wet weight (FIG. 8B), an activity
that tracked with the dose dependent downregulation of ESR1
expression (FIG. 8C). Notably, no decrease in ESR1 expression was
observed in animals treated with 0.3 mg/kg, the dose where the
agonist activity of RAD1901 in the uterine wet weight assay was
observed (FIG. 8C). As observed in clinical trial for hot flashes,
RAD1901 exhibited a complex biphasic pharmacology that manifests as
antagonist activity at the higher doses.
Example 10
[0161] RAD1901 Exhibits Biphasic Activity with Respect to
ESR1-Dependent Tumor Growth
[0162] As shown above, high dose RAD1901 (20 mg/kg) inhibited the
growth of ESR1-dependent MCF7-cell derived tumors in mice. However,
the observation that uterine wet weight was increased in mice
treated with doses of RAD1901 that were lower than that required to
effect ESR1 turnover highlighted the need to examine whether a
similar biphasic pharmacology was manifest in breast tumors. To
further evaluate the pharmacology of RAD1901, a second xenograft
tumor study was conducted in which MCF7 tumors were established
under estrogen stimulation. For FIG. 9A, MCF7 xenograft tumors were
initiated in ovariectomized female nu/nu mice as in FIG. 5.
Estrogen pellets were surgically removed when tumors reached
.about.0.1 cm.sup.3 volume, and animals (n=6-10) then received
daily treatment with vehicle or RAD1901 (RAD), (0.3-10 mg/kg sc).
Mean tumor volume.+-.SEM per day of treatment was presented.
Significance as compared to the vehicle (2-way ANOVA of matched
values followed by Bonferroni comparison) was indicated
(*p<0.05, **p<0.0005). For FIG. 9B, uterine wet weight at
sacrifice (measured as in FIG. 8) and % change in tumor volume (as
compared to size at randomization) calculated using the final
measurement recorded for mice in (FIG. 9A) are graphically
presented. For FIGS. 9C-9D, expression of ESR1 target genes in
tumors was analyzed essentially as in FIG. 5. Estrogen only samples
from FIG. 5D were included for comparison. For FIG. 9E, ESR1 levels
in tumor tissues were analyzed as in FIG. 8 and were normalized to
similarly detected Lamin-A. Significant downregulation (*p<0.05)
was determined by ANOVA followed by Bonferroni comparison.
[0163] When tumors reached .about.0.1 cm.sup.3 volume, estrogen was
discontinued and animals were randomized to treatment with vehicle
or RAD1901 (0.3, 1, 3, or 10 mg/kg). As observed in the uterine
weight assay, RAD1901 exhibited a biphasic response, in that
significant stimulation of tumor growth was observed in animals
treated with 1 or 3 mg/kg RAD1901 that was not apparent at the
higher dose (FIG. 9A). Although certainly less than that which was
observed following E2 stimulation (FIG. 5E), the increased tumor
volume in the 1 and 3 mg/kg groups was significant and of similar
magnitude to that for partial ESR1 agonists. Interestingly, an
evaluation of the final tumor size and uterine wet weight of these
animals at sacrifice revealed that the pharmacology of RAD1901 was
affected by both dose and tissue. Specifically, as reported above
in a different strain of mice, stimulation of uterine weight was
apparent in mice treated with only the 0.3 mg/kg dose of RAD1901,
while tumor size was significantly increased in 1 or 3 mg/kg
RAD1901 groups (FIG. 9B). Whereas the expression of classical ESR1
target genes such as PGR was not observed in the tumors in the 1 or
3 mg/kg treatment groups (FIG. 9C), the expression of AGR2 (FIG.
9D) and others SERM-regulated genes associated with tamoxifen
resistance (not shown) was observed. Immunoblot analysis of tumor
extracts revealed a dose-dependent downregulation of ESR1 by
RAD1901 that reflected its actions as an inhibitor of tumor growth
(FIG. 9E). Together these data highlight the complex pharmacology
of RAD1901 and stress the importance of developing biomarkers that
read on the partial agonist activity of the drug and which can be
used for dose optimization in clinical studies.
Example 11
[0164] Elucidation of the Mechanisms that Distinguish RAD1901 from
Other SERDs
[0165] Whether the dose-dependent agonist/antagonist activity of
RAD1901 could be observed at the level of target gene regulation
was determined. Specific sets of ESR1 target genes whose expression
was differentially regulated by estrogens and SERMs and which could
be used to distinguish between different SERMs and SERDs have been
identified. A subset of these genes, those regulated by (a)
agonists (with no or minimal response to SERMs), (b) SERMs (with no
or little response to E2), or (c) either agonists or SERMs, were
selected to profile RAD1901 activity.
[0166] MCF7 cells were treated for 24 hours with 10.sup.-7 M
vehicle (Veh), ICI, 40HT, raloxifene (Ralox), E2 (10.sup.-9 M) or
RAD1901 (10.sup.-9-10.sup.-6 M). The expression of ESR1 target
genes responsive to (FIG. 10A) agonists, (FIG. 10B) primarily
SERMs, or (FIG. 10C) SERMs and agonists was analyzed as in FIG. 5.
Relative changes of these and additional target genes designed to
evaluate dose dependent response to RAD1901 are presented in FIG.
11. Significant target gene regulation (*p<0.05) as compared to
the vehicle control was detected by 2-way ANOVA followed by
Fisher's LSD (Graphpad Prism 6). FIG. 10D shows the interaction
between ESR1 and conformation-specific peptides in a mammalian
two-hybrid system. Triplicate wells of SKBR3 cells were transfected
with plasmids expressing ESR1 fused to VP16 together with Gal4DBD
alone (control) or Gal4DBD fused to ESR1 interacting peptides noted
on the horizontal axis. Cells were then treated with the indicated
ESR1 ligands (10.sup.-7 M unless otherwise indicated). Interaction
of ESR1 with the Gal4DBD peptide constructs was detected through
activation of a Gal4-responsive luciferase reporter construct and
was normalized to detected .beta.-galactosidase activity expressed
in a constitutive manner using a second vector. Normalized response
was expressed as fold increase over the detected level of
interaction between Gal4DBD alone and ESR1-VP16 in the absence of
ligand (Veh). For FIG. 10E, the effect of SERMs and SERDs
(10.sup.-6 M) on the proliferation of MCF7 cells in response to E2
(10.sup.-9 M) or insulin (2.times.10.sup.-9 M) was evaluated as in
FIG. 5. Statistically similar treatments (2-way ANOVA followed by
Bonferroni comparison) are indicated by letters. For FIG. 11, MCF7
cells were treated for 24 hours with 10.sup.-7 M vehicle (Veh),
ICI, 40HT, raloxifene (Ralox), E2 (10.sup.-9 M) or RAD1901
(10.sup.-9-10.sup.-6 M). The expression of ESR1 target genes
responsive to (A) agonists, (B) primarily SERMs, or (C) SERMs and
agonists was analyzed using RT qPCR following RNA isolation. mRNA
expression was normalized (.DELTA..DELTA.CT analysis) to the
similarly detected 36B4 housekeeping. The profiles presented in
dendogram format were analyzed with the Ward hierarchical
clustering algorithm (JMP 11) using standardized data.
[0167] Reflecting the pharmacology observed in vivo, the expression
pattern of these target genes exhibited a biphasic response to
RAD1901 with agonist activity been observed at lower doses and more
complete antagonist activity apparent at higher doses (FIGS.
10A-10C and FIG. 11). These data confirmed the unique pharmacology
of RAD1901 and suggested that this drug may function by a mechanism
that was distinct from other ESR1 downregulators.
[0168] The pharmacology of ESR1 ligands reflects their influence on
the overall structure of the receptor and on the impact which this
has on coregulator recruitment. Given the distinct pharmacology
exhibited by RAD1901, it may enable ESR1 to adopt a unique
conformation. A conformational profiling tool was used to
interrogate the ESR1-RAD1901 complex. In this assay a modified
two-hybrid assay was used to assess the binding of a series of
short peptides that survey the protein-protein interaction surfaces
on ESR1 that were presented when occupied by different ligands. As
shown in FIG. 10D, the interaction profile observed in the presence
of RAD1901 was completely distinct from any other known ligand.
Notable was the ability of RAD1901 to disengage the classical
coregulator-binding surface (AF2) as indicated by the decreased
interaction with the peptides that report on "agonist activity"
(ASC1 and PGC1). No interaction was observed with peptides that
report on the ESR1 structures adopted upon binding tamoxifen
(.alpha..beta.V) or ICI (.beta.I2). There was significant
interaction of all in the presence of RAD1901, a peptide that
reports on a cryptic protein-protein interaction surface in the
hinge region of the receptor. RAD1901 enabled ESR1 to assume a
unique conformation that was distinct from that apparent upon
binding agonists, the SERM tamoxifen, and other known SERDs (FIG.
10D).
[0169] In addition to classical agonists, ESR1 transcriptional
activity can also be induced by treating cells with growth factors
such as EGF, IGF1 or insulin. Whereas the mechanisms underlying
this "ligand-independent" activity were likely to be complex, this
alternate pathway of activation contributes to resistance to
endocrine therapy in breast cancer. The comparative pharmacology of
RAD1901 was evaluated for its ability to suppress growth factor
dependent activation of ESR1. In this assay, it was determined that
although all of the SERMs and SERDs tested inhibited E2-stimulated
proliferation, only BZA and ICI efficiently inhibited
insulin-stimulated proliferation, while 40HT, Ralox, and RAD1901
were without effect (FIG. 10E). These results confirm the unique
mechanism of action of RAD1901. RAD1901 may be delivered in
combination with another drug that inhibits ligand-independent
activation of ESR1 to achieve maximal clinical response.
[0170] It is understood that the foregoing detailed description and
accompanying examples are merely illustrative and are not to be
taken as limitations upon the scope of the invention, which is
defined solely by the appended claims and their equivalents.
[0171] Various changes and modifications to the disclosed
embodiments will be apparent to those skilled in the art. Such
changes and modifications, including without limitation those
relating to the chemical structures, substituents, derivatives,
intermediates, syntheses, compositions, formulations, or methods of
use of the invention, may be made without departing from the spirit
and scope thereof.
[0172] For reasons of completeness, various aspects of the
invention are set out in the following numbered clauses:
[0173] Clause 1. A method of treating estrogen receptor positive
cancers of the brain in a subject, the method comprising
administering a compound represented by the following formula
I:
##STR00013##
wherein [0174] TZ represents a C.sub.1-C.sub.4 alkylene group or
--CR.sup.f'R.sup.g'--CH.sub.2--O-- wherein R.sup.f' and R.sup.g'
independently represent hydrogen or a C.sub.1-C.sub.6 alkyl group;
[0175] A represents a 5- to 14-membered heteroarylene group which
may have a substituent or a C.sub.6-C.sub.14 arylene group which
may have a substituent; [0176] Y represents --CH.sub.2--NR.sup.c--
wherein R.sup.c represents hydrogen or a C.sub.1-C.sub.6 alkyl
group which may have a substituent; [0177] ring G represents the
following formula:
[0177] ##STR00014## [0178] R' represents 1 to 4 substituents
independently selected from a hydrogen atom, a C.sub.1-C.sub.6
alkoxy group, and a hydroxyl group; [0179] a partial structure in
formula (I) represented by the following formula:
[0179] ##STR00015## [0180] R'' represents hydrogen, a hydroxyl
group that may be further protected by a protecting group or a
C.sub.1-C.sub.6 alkoxy group which may have a substituent; and
[0181] R.sup.a and R.sup.b are the same as or different from each
other and each represents a hydrogen atom, a C.sub.1-C.sub.6 alkyl
group which may have a substituent, or a C.sub.3-C.sub.8 cycloalkyl
group which may have a substituent, or when R.sup.a and R.sup.b are
bonded together, they may form, together with the nitrogen atom
that is adjacent to R.sup.a and R.sup.b, a 4- to 10-membered single
ring which may have a substituent; and [0182] L represents a single
bond, or a salt thereof.
[0183] Clause 2. The method of clause 1, wherein the cancer is
Breast cancer brain metastases, Astrocytoma, Atypical Teratoid
Rhabdoid Tumor (ATRT), Chondrosarcoma, Choroid Plexus Carcinoma,
Craniopharyngioma, Ependymoma, Germ Cell Tumor, Glioblastoma,
Glioma, Hemangioma, Juvenile Pilocytic Astrocytoma,
Medulloblastoma, Meningioma, Neurofibroma, Neuronal and Mixed
Neuronal-Glial Tumors, Oligoastrocytoma, Oligodendroglioma, Pineal
Tumor, Pituitary Tumor, PNET--(primitive neuroectodermal tumor),
Schwannoma, and Leptomeningeal metastases.
[0184] Clause 3. A method of treating breast cancer brain
metastasis in a subject, the method comprising administering a
compound represented by the following formula I:
##STR00016##
wherein [0185] TZ represents a C.sub.1-C.sub.4 alkylene group or
--CR.sup.f'R.sup.g'--CH.sub.2--O-- wherein R.sup.f' and R.sup.g'
independently represent hydrogen or a C.sub.1-C.sub.6 alkyl group;
[0186] A represents a 5- to 14-membered heteroarylene group which
may have a substituent or a C.sub.6-C.sub.14 arylene group which
may have a substituent; [0187] Y represents --CH.sub.2--NR.sup.c--
wherein R.sup.c represents hydrogen or a C.sub.1-C.sub.6 alkyl
group which may have a substituent; [0188] ring G represents the
following formula:
[0188] ##STR00017## [0189] R' represents 1 to 4 substituents
independently selected from a hydrogen atom, a C.sub.1-C.sub.6
alkoxy group, and a hydroxyl group; [0190] a partial structure in
formula (I) represented by the following formula:
[0190] ##STR00018## [0191] R'' represents hydrogen, a hydroxyl
group that may be further protected by a protecting group or a
C.sub.1-C.sub.6 alkoxy group which may have a substituent; and
[0192] R.sup.a and R.sup.b are the same as or different from each
other and each represents a hydrogen atom, a C.sub.1-C.sub.6 alkyl
group which may have a substituent, or a C.sub.3-C.sub.8 cycloalkyl
group which may have a substituent, or when R.sup.a and R.sup.b are
bonded together, they may form, together with the nitrogen atom
that is adjacent to R.sup.a and R.sup.b, a 4- to 10-membered single
ring which may have a substituent; and [0193] L represents a single
bond, or a salt thereof.
[0194] Clause 4. A method of treating a cancer in a subject,
wherein the cancer is resistant to an estrogen receptor modulator,
the method comprising administering a compound represented by the
following formula I:
##STR00019##
wherein [0195] TZ represents a C.sub.1-C.sub.4 alkylene group or
--CR.sup.f'R.sup.g'--CH.sub.2--O-- wherein R.sup.f' and R.sup.g'
independently represent hydrogen or a C.sub.1-C.sub.6 alkyl group;
[0196] A represents a 5- to 14-membered heteroarylene group which
may have a substituent or a C.sub.6-C.sub.14 arylene group which
may have a substituent; [0197] Y represents --CH.sub.2--NR.sup.c--
wherein R.sup.c represents hydrogen or a C.sub.1-C.sub.6 alkyl
group which may have a substituent; [0198] ring G represents the
following formula:
[0198] ##STR00020## [0199] R' represents 1 to 4 substituents
independently selected from a hydrogen atom, a C.sub.1-C.sub.6
alkoxy group, and a hydroxyl group; [0200] a partial structure in
formula (I) represented by the following formula:
[0200] ##STR00021## [0201] R'' represents hydrogen, a hydroxyl
group that may be further protected by a protecting group or a
C.sub.1-C.sub.6 alkoxy group which may have a substituent; and
[0202] R.sup.a and R.sup.b are the same as or different from each
other and each represents a hydrogen atom, a C.sub.1-C.sub.6 alkyl
group which may have a substituent, or a C.sub.3-C.sub.8 cycloalkyl
group which may have a substituent, or when R.sup.a and R.sup.b are
bonded together, they may form, together with the nitrogen atom
that is adjacent to R.sup.a and R.sup.b, a 4- to 10-membered single
ring which may have a substituent; and [0203] L represents a single
bond, or a salt thereof.
[0204] Clause 5. The method of clause 4, wherein the cancer is de
novo resistant to the estrogen receptor modulator.
[0205] Clause 6. The method of clause 4, wherein the resistance to
the estrogen receptor modulator is acquired.
[0206] Clause 7. The method of clause 4, wherein the estrogen
receptor modulator is a selective estrogen receptor modulator
(SERM).
[0207] Clause 8. The method of clause 7, wherein the SERM is
tamoxifen, idoxifene, raloxifene or ICI 182,780.
[0208] Clause 9. The method of any one of clauses 4-8, wherein the
cancer is breast, endometrial or ovarian cancer.
[0209] Clause 10. The method of any one of clauses 4-9, wherein the
cancer is breast cancer.
[0210] Clause 11. The method of any one of clauses 1-10, wherein
R.sup.a and R.sup.b independently represent a hydrogen atom, a
methyl group, an ethyl group, a n-propyl group, an iso-propyl
group, a n-butyl group, an iso-butyl group, or a tert-butyl
group.
[0211] Clause 12. The method of any one of clauses 1-10, wherein
-T-Z-- represents --CH.sub.2CH.sub.2-- or
--C(CH.sub.3).sub.2CH.sub.2O--.
[0212] Clause 13. The method of any one of clauses 1-10, wherein Y
represents --CH.sub.2--N(CH.sub.2CH.sub.3)-- or
--CH.sub.2--N(CH.sub.2CH.sub.2OH)--.
[0213] Clause 14. The method of any one of clauses 1-10, wherein
each of R'' independently represents a hydrogen atom or a methoxy
group.
[0214] Clause 15. The method of any one of clauses 1-10, wherein
R'' represents a hydroxyl group.
[0215] Clause 16. The method of any one of clauses 1-10, wherein A
represents a phenylene group.
[0216] Clause 17. The method of any one of clauses 1-10, wherein
the compound is
(R)-6-{2-{ethyl[4-(2-ethylaminoethyl)benzyl]amino}-4-methoxyphenyl}-5,6,7-
,8-tetrahydronaphthalen-2-ol.
[0217] Clause 18. The method of any one of clauses 1-10, wherein an
effective amount of the compound is administered.
[0218] Clause 19. The method of any one of clauses 1-10, wherein
the effective amount comprises a high dosage.
[0219] Clause 20. The method of clause 19, wherein the high dosage
is more than about 20 mg/kg.
[0220] Clause 21. The method of clause 19 or 20, wherein the high
dosage is about 20 mg/kg to about 100 mg/kg.
[0221] Clause 22. The method of any one of clauses 1-10, wherein
the compound is administered by oral administration, intravenous
administration, intradermal injection, intramuscular injection, or
subcutaneous injection.
[0222] Clause 23. The method of any one of clauses 1-10, further
comprising administering an effective amount of at least one
compound selected from the group consisting of a cyclin-dependent
kinase 4 and 6 inhibitor (CDK4/6 inhibitor), an antiestrogen, a
ligand of retinoic acid or retinoxic X receptor, an antiprogestin,
an antiandrogen, vitamin D or metabolite thereof, a farnesyl
transferase inhibitor, a PPAR.alpha. or gamma agonist and a MAP
kinase inhibitor.
* * * * *